TaqMan® hPSC Scorecard™ Panel
Contents
1. Product Quantity Catalog no Dulbecco s Modified Eagle Medium DMEM with GlutaMAX I high glucose Don FUSION DMEM F 12 with GlutaMAX I 500 mL 10565 018 mm 100 mL 10828 010 KnockOut Serum Replacement 500 mL 10828 028 Essential 8 Medium Prototype 50X 500 mL A14666SA StemPro hESC SFM Kit for 500 mL of complete StemPro hESC SFM LR PHISUD BE MEM Non Essential Amino Acids Solution 100 mL 11140 050 10 mM Basic Fibroblast Growth Factor bFGF 10 pg PHG0264 recombinant human Bovine Albumin Fraction V Solution BSA 7 5 100 mL 15260 037 Fetal Bovine Serum FBS ES Cell Qualified 500 mL 16141 079 p Mercaptoethanol 1000X liquid 50 mL 21985 023 DPBS no Calcium no Magnesium 500 mL 14190 144 For more information about the following products refer to our website at www lifetechnologies com or contact Technical Support page 43 Product Quantity Catalog no rm e wu imb e Attachment Factor 100 mL 5 006 100 UltraPure 0 5 M EDTA pH 8 0 4 x 100 mL 15575 020 0 05 Trypsin EDTA 1X Phenol Red 100 mL 25300 054 Collagenase Type IV powder 1g 17104 019 For more information about the following products refer to our website at www lifetechnologies com or contact Technical Support page 43 Product Quantity Catalog no Countess Automated Cell Counter 1 unit C10227 TM StemPro EZPassage Disposable Stem Cell 10 units 23181 010 Passaging Tool2. 39 Chemical Safety Chemical Hazard Warning General Safety Guidelines 40 Appendix E Safety WARNING CHEMICAL HAZARD Before handling any chemicals refer to the Safety Data Sheet SDS provided by the manufacturer and observe all relevant precautions i WARNING CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument T WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles To minimize the hazards of chemicals Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See Safety Data Sheets SDS page 43 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For
3. In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 www cdc gov OD ohs biosfty bmbl4 bmbl4toc htm Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check your local guidelines and legislation on biohazard and biosafety precaution and the best practices published in the World Health Organisation WHO Laboratory Biosafety Manual third edition www who int csr resources publications biosafety WHO CDS CSR LYO 2004 11 en Documentation and Support Technical Support Obtaining support Safety data sheets SDS Certificate of analysis Limited product warranty For the latest services and support information for all locations go to www lifetechnologies com At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support techsupport lifetech com e Search for user documents SDSs vector maps and sequences application notes formulat
4. TagMan hPSC Scorecard 384w The TaqMan hPSC Scorecard Panel 384w Cat no A15870 and the TaqMan hPSC Scorecard Kit 384w Cat no A15872 contain the components listed below Each 384 well plate can be used to analyze four cDNA samples Catalog no Component Composition Amount A15870 15872 TagMan hPSC i i Scorecard Panel 384w TaqMan probes in a 384 well plate 1 plate v v MicroAmp Optical Adhesive Film Optical adhesive covers 1 each Y v Solution containing AmpliTaq Gold DNA TaqMan Gene Polymerase UP Ultra Pure Uracil DNA 5mL Expression Master Mix Glycosylase dNTPs with dUTP Passive Reference 1 and optimized mix components TagMan hPSC TaqMan hPSC Scorecard Panel Quick EA E a Scorecard Panel ORC Reference Card TaqMan Gene Expression Master Mix Cat no 4369016 is also available separately from Life Technologies see page 38 Continued on next page Kit Contents and Storage continued TaaMan hPSC The TagMan hPSC Scorecard Panel 2 x 96w FAST Cat no A15876 and the q 4 TM Scorecard 2 x 96w TaqMan hPSC Scorecard Kit 2 x 96w FAST Cat no A15871 contain the FAST components listed below Each 96 well plate can be used to analyze one cDNA sample Catalog no Component Composition Amount A15876 A15871 TagMan hPSC Scorecard Panel 96w TaqMan probes in a 96 well plate 2 plates v v FAST asi cu E Optical Adhesive Covers 2 e
5. Panel 96w FAST are MicroAmp Optical 96 or 384 well Reaction Plates standard or Fast that contain dried down TaqMan Gene Expression Assays specifically formulated for evaluating human embryonic stem cells ESC and human induced pluripotent stem cells iPSC to confirm their pluripotency and to predict their differentiation potential and outcome The gene expression assays contain a collection of predesigned gene specific primer and probe sets for performing quantitative gene expression studies on the cDNA samples prepared from undifferentiated or differentiated human ESCs and iPSCs For more information on TaqMan Gene Expression Assays see Appendix C Background Information on page 37 After isolating total RNA from human ESC or iPSC cultures and using it to generate cDNA in a reverse transcription RT reaction TaqMan hPSC Scorecard Panels and associated reagents are used to quantitate RNA expression levels of genetic markers for pluripotency and differentiation potential as well as endogenous controls The gene expression data are then analyzed using the web based hPSC Scorecard Analysis Software for the pluripotency and differentiation potential of the cells from which the total RNA is isolated To do this you e Isolate total RNA from human ESCs or iPSCs by TRIzol organic phase extraction or other preferred method e Prepare each cDNA sample by performing eight reverse transcription reactions per total RNA sample e C
6. 100 mm dish 60 cm 6 0 mL 10 0 mL 10 Undifferentiated Cells Feeder Free Culture in StemPro hESC SFM Introduction Materials needed Prepare media reagents and matrix coated culture vessels Culture and harvest cells This section provides instructions on harvesting undifferentiated ESCs or iPSCs maintained as feeder free cultures on Geltrex matrix coated vessels in complete StemPro hESC medium for total RNA extraction You will need to harvest at least 5 x 10 cells per sample to isolate sufficient total RNA for the subsequent steps of the workflow e DMEM F 12 with GlutaMAX I Cat no 10565 018 e StemPro hESC SFM Kit Cat no A1000701 e p Mercaptoethanol 1000X liquid Cat no 21985 023 e Basic Fibroblast Growth Factor bFGF recombinant human Cat no PGH0264 e Collagenase Type IV Cat no 17104 019 e DPBS no Calcium no Magnesium Cat no 14190 144 e Geltrex hESC qualified Cat no A1413302 e TRIzol Reagent Cat no 18596 018 e Cell Scraper Falcon Cat no 353085 e Appropriate tissue culture plates and supplies The following media reagents and culture vessels are needed for culturing and harvesting undifferentiated ESCs or iPSCs maintained as feeder free cultures in complete StemPro hESC medium For instructions on preparing the media and reagents listed below see Appendix A Recipes page 28 For instructions on coating culture vessels with Geltrex
7. Appendice E Salt leo n oscorimeuosu imina anaa udbes suce estesa iud enc o conus decet pe losuaut a 40 Chemical Saf ty z enano telee ttti ee drapeau dien er rente 40 Chemical Waste Safety ete cette eec ciment tir rt eiae ip ne aee pendet 41 Biological Hazard Safety et ete eee idee E ehe ne e di recepere Red 42 Documentation and SUpDOTE scisco pat eere x NER UR IU E RUE cp pi d RERO RD E HAMM pria UN 43 Technical S ppotIsu ance oe nibo aerei iege itii tam arid kava ial P ee cre heat 43 Preface About This Guide Purpose of this guide User attention words Safety alert words This user guide is provides detailed procedures and reference materials for generating and preparing samples from undifferentiated and differentiated ESCs and iPSCs and for analyzing these samples using the TaqMan hPSC Scorecard Panels for pluripotency and differentiation potential This user guide does not provide instructions for the hPSC Scorecard Analysis Software Go to www lifetechnologies com scorecardsoftwaremanual for detailed instructions on using the software To access the hPSC Scorecard Analysis Software go to www lifetechnologies com scorecarddata Two user attention words appear in Life Technologies user documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product n IMPORTANT
8. Kit 384w 1 kit A15872 TaqMan hPSC Scorecard Panel 2 x 96w FAST 2 plates A15876 TaqMan hPSC Scorecard Kit 2 x 96w FAST 1 kit A15871 25 covers 4360954 MicroAmp Optical Adhesive Film 100 covers 4311971 TaqMan Gene Fxpression Master Mix iPack Lx mi 200 reactions 4369016 TaqMan Fast Advanced Master Mix s Im 100 reactions 4444556 Accessory products For more information about the following products refer to our website at www lifetechnologies com or contact Technical Support page 43 Product Quantity Catalog no High Capacity cDNA Reverse Transcription 200 reactions 4374966 Kit with RNase Inhibitor 1000 reactions 4374967 100 mL 15596 026 ine Rese 200 mL 15596 018 DNA free Kit 50 reactions AM1906 UltraPure DNase RNase Free Distilled 500 mL 10977 015 Water 10 x 500 mL 10977 023 Cells For more information about the following products refer to our website at www lifetechnologies com or contact Technical Support page 43 Product Quantity Catalog no Gibco Mouse Embryonic Fibroblasts inradiated i x W cells ei Ab PAE Continued on next page 38 Accessory Products continued Media sera and supplements Matrices and dissociation reagents Equipment For more information about the following products refer to our website at www lifetechnologies com or contact Technical Support page 43
9. matrix see Appendix B Preparing Culture Vessels page 34 e StemPro hESC SFM medium e StemPro wash solution e 10X Collagenase IV Solution 10 mg mL e Geltrex matrix coated culture vessels 1 Coatthe culture vessels with Geltrex matrix at least 1 hour before passaging the cells Warm the appropriate amount of 10 mg mL 10X Collagenase IV solution StemPro wash solution and complete StemPro hESC SFM medium to 37 C in a water bath 2 When the ESC or iPSC culture has reached the desired confluency to yield at least 5 x 10 cells per sample aspirate off the cell culture medium i e StemPro hESC SFM 3 Gently add 10 mg mL Collagenase Type IV 10X solution to the culture vessel containing ESCs or iPSCs Adjust the volume of Collagenase IV for various dish sizes refer to Table 2 page 13 Continued on next page 11 Undifferentiated Cells Feeder Free Culture in StemPro hESC SFM continued Culture and harvest 4 Incubate for 3 5 minutes in a 37 C 5 CO incubator until the edges of the cells continued colonies begin to curl Note Incubation times may vary among different batches of collagenase therefore you need to determine the appropriate incubation time by examining the colonies Note that the required exposure time shortens as the enzymatic activity of collagenase increases i e 200 000 U mg at 1 mg mL concentration will take longer than stocks of 290 000 U mg at 1 mg mL concentrat
10. medium MEF CM The final volume of the MEF CM depends on the vessel used refer to Table 2 page 10 for the final volume of medium required for each vessel Use a typical split ratio of 1 3 to 1 5 per plate depending on amount of cell clusters Continued on next page Undifferentiated Cells Feeder Dependent Culture continued Culture and harvest 12 Move the culture vessel in several quick short back and forth and side to side motions to disperse the cells evenly across its surface and then return cells continued the vessel to the incubator 13 Continue to culture the cells until they are 80 90 confluent changing the spent MEF CM daily 14 On the day of harvesting aspirate the culture medium and wash the cells once with 5 mL of DPBS for 2 3 minutes 15 Aspirate the DPBS and discard 16 Add 1 mL of TRIzol reagent and incubate for 2 3 minutes Scrape the plate with a sterile cell scraper and collect the slurry into a sterile RNAse free microcentrifuge tube 17 Store the cells at 80 C until ready for RNA isolation Table 1 Volume of Collagenase IV 1 mg mL and MEF CM required Culture Vessel Surface Area Volume of Collagenase IV Volume of MEF CM 6 well plate 10 cm well 1 0 mL per well 2 0 mL per well 12 well plate 4 cm well 0 5 mL per well 1 0 mL per well 24 well plate 2 cm well 0 25 mL per well 0 5 mL per well 35 mm dish 10 cm 1 0 mL 2 0 mL 60 mm dish 20 cm 2 0 mL 4 0 mL
11. minutes in a 37 C 5 CO incubator until the edges of the calls continued colonies begin to curl Note Incubation times may vary among different batches of collagenase therefore you need to determine the appropriate incubation time by examining the colonies Note that the required exposure time shortens as the enzymatic activity of collagenase increases i e 200 000 U mg at 1 mg mL concentration will take longer than stocks of 290 000 U mg at 1 mg mL concentration 6 When the edges of the colonies are starting to pull away from the vessel carefully aspirate the Collagenase IV solution from the vessel without disturbing the attached cell layer and gently rinse the cells with 3 mL of pre warmed DMEM F 12 solution 7 Aspirate the wash solution and add the appropriate volume of pre warmed complete Essential 8 medium refer to Table 3 page 16 8 Usea sterile cell scraper to gently remove the cell clumps Pipette the cell suspension across the plate surface with a 5 mL pipette to break up the detached colonies into smaller clumps Do not create a single cell suspension usually 3 5 pipetting motions will suffice to dislodge and resuspend the cells 9 Gently transfer the cell clumps into a 15 mL tube Depending on the culture vessel use an additional 1 3 mL of pre warmed complete StemPro hESC SFM medium to collect any cell clumps remaining in the vessel and add to the 15 mL conical tube 10 Centrifuge cell suspen
12. the outside of the vial with 70 ethanol before placing it in the cell culture hood Pipet the thawed cells gently into a 15 mL conical tube Rinse the cryovial with 1 mL of pre warmed MEF medium see page 31 Transfer the medium to the same 15 mL tube containing the cells Add 4 mL of pre warmed MEF medium dropwise to the cells Gently mix by pipetting up and down Note Adding the medium slowly helps the cells to avoid osmotic shock Centrifuge the cells at 200 x g for 5 minutes Aspirate the supernatant and resuspend the cell pellet in 5 mL of pre warmed MEF medium Remove 20 uL of the cell suspension and determine the viable cell count using your method of choice e g Countess Automated Cell Counter Continued on next page 35 MEF Culture Dishes continued Plating MEFs Centrifuge the remaining cell suspension step 9 previous page at 200 x g for 5 minutes at room temperature Aspirate the supernatant Resuspend the cell pellet in MEF medium see page 31 to a density of 2 5 x 10 cells mL Aspirate the gelatin solution from the gelatin coated culture vessel Add the appropriate amount of MEF medium into each culture vessel refer to the table below Into each of these culture vessels add the appropriate amount of MEF suspension refer to the table below Note The recommended plating density for Gibco Mouse Embryonic Fibroblasts Irradiated Cat no 1520 100 is 2 5 x 10 cells cm 6 Move the
13. vessels are needed for culturing and harvesting undifferentiated feeder dependent ESCs or iPSCs For instructions on preparing the media and reagents listed below see Appendix A Recipes page 28 For instructions on coating culture vessels with Geltrex matrix see Appendix B Preparing Culture Vessels page 34 e MEF medium e ESC medium e MEF conditioned medium MEF CM e 1X Collagenase IV Solution 1 mg mL e Geltrex matrix coated culture vessels Continued on next page Undifferentiated Cells Feeder Dependent Culture continued Culture and harvest cells 1 10 11 Culture ESCs or iPSCs on inactivated MEF feeder cells using complete ESC medium When the ESC or iPSC culture has reached the desired confluency to yield at least 5 x 10 cells per sample aspirate off the cell culture medium i e ESC medium Add Collagenase Type IV 1 mg mL solution to the dish containing ESCs or iPSCs Adjust the volume of Collagenase IV for various dish sizes refer to Table 1 page 10 Incubate for 30 45 minutes in a 37 C 5 CO incubator N Note Incubation times may vary among different batches of E collagenase therefore you need to determine the appropriate incubation time by examining the colonies Note that the required exposure time shortens as the enzymatic activity of collagenase increases i e 200 000 U mg at 1 mg mL concentration will take longer than stocks of 290 000 U mg at 1 mg mL con
14. 0 C until ready for RNA isolation Table 2 Volume of Collagenase IV 10 mg mL and complete StemPro hESC SFM medium required Culture Vessel Surface Area Volume of Collagenase IV Volume of complete StemPro hESC SFM 6 well plate 10 cm well 1 0 mL per well 2 0 mL per well 12 well plate 4 cm well 0 5 mL per well 1 0 mL per well 24 well plate 2 cm well 0 25 mL per well 0 5 mL per well 35 mm dish 10 cm 1 0 mL 2 0 mL 60 mm dish 20 cm 2 0 mL 4 0 mL 100 mm dish 60 cm 6 0 mL 10 0 mL 13 Undifferentiated Cells Feeder Free Culture in Essential 8 Medium Introduction Materials needed Prepare media reagents and matrix coated culture vessels Culture and harvest cells 14 This section provides instructions on harvesting undifferentiated ESCs or iPSCs maintained as feeder free cultures on Geltrex matrix coated vessels in complete Essential 8 medium for total RNA extraction You will need to harvest at least 5 x 10 cells per sample to isolate sufficient total RNA for the subsequent steps of the workflow e Essential 8 Medium prototype Cat no A14666SA e DMEM F 12 with GlutaMAX I Cat no 10565 018 e Collagenase Type IV Cat no 17104 019 e DPBS no Calcium no Magnesium Cat no 14190 144 e Geltrex hESC qualified Cat no A1413302 e TRIzol Reagent Cat no 18596 018 e Cell Scraper Falcon Cat no 353085 e Appropriate tissue cultur
15. B medium If necessary add more EB medium into the 15 mL tube to bring up the volume to 5 mL Note If using a different size vessel add more pre warmed EB medium into the 15 mL tube to bring up the volume of the cell suspension to the appropriate level for the vessel used refer to Table 4 below Generally use one plate of ESCs or iPSCs per one plate of EBs to be formed Place Petri dish containing the cells in a 37 C 5 CO incubator to allow the cells to form EBs The next day and every other day thereafter replace the spent medium with fresh pre warmed EB medium To replace the medium gently transfer the EB suspension into a 15 mL conical tube and allow the EBs to sediment down by gravity for 10 15 minutes Then gently aspirate off the supernatant and re suspend the EBs with fresh pre warmed EB medium Return the EB suspension into the same culture vessel for continued growth On day 7 of incubation harvest the cells as described in Harvest cells Option 1 7 day EB suspension page 19 N Note Culturing EBs in suspension for 7 days is usually sufficient for the analysis of random differentiation If extended culture is desired use the optional protocol for adherent EB culture as described in Harvest cells Option 2 4 day EB suspension and adherent culture page 19 Table 4 Volume of EB medium required Final volume of cell Culture Vessel Surface Area suspension in EB medium 6 w
16. Panel 384w must be run on RT PCR systems with standard thermal cycling blocks Compatible Applied Biosystems TaqMan hPSC Scorecard Panel RT PCR systems TaqMan hPSC Scorecard Panel 384w e QuantStudio 12K Flex System with 384 well Block e ViiA 7 Real Time PCR System with 384 well Block TaqMan hPSC Scorecard Panel 96w FAST e StepOnePlus Real Time PCR System e ViiA 7 Real Time PCR System with Fast 96 well Block TaqMan hPSC Scorecard Panel Workflow Experiment outline The table below describes the major steps needed to generate and prepare cDNA samples for analysis using the TaqMan hPSC Scorecard Panels For more details refer to the pages indicated Step Action Page 1 Harvest ESCs or iPSCs from Undifferentiated cells feeder dependent culture 8 Undifferentiated cells feeder free culture in StemPro hESC SFM 11 Undifferentiated cells feeder free culture in Essential 8 medium 14 Randomly differentiated cells i e embryoid bodies 17 2 Isolate total RNA from cells 20 3 Optional Remove contaminating DNA from RNA sample 22 4 Quantitate total RNA and asses its quality 25 5 Generate cDNA by reverse transcription 26 6 Perform TagMan qRT PCR 28 7 Analyze data using the hPSC Scorecard Analysis Software 30 Sample Generation Undifferentiated Cells Feeder Dependent Culture Introduction Materials needed Prepare media reagents and matrix coat
17. Provides information that is necessary for proper instrument operation accurate installation or safe use of a chemical Four safety alert words appear in Life Technologies user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Provides information that is necessary for proper instrument operation accurate installation or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations PP BS Product Information Kit Contents and Storage Types of kits This manual is supplied with the products listed below For a list of components supplied with each catalog number see below Product Catalog no TaqMan hPSC Scorecard Panel 384w 415870 TaqMan hPSC Scorecard Kit 384w A15872 TaqMan hPSC Scorecard Panel 2 x 96w FAST A15876 TaqMan hPSC Scorecard Kit 2 x 96w FAST A15871
18. USER GUIDE applied biosystems by Kafe technologies TagMan hPSC Scorecard Panel For rapid confirmation of pluripotency and prediction of differentiation potential Catalog Numbers A15870 A15871 A15872 A15876 Publication Number MAN0008384 Revision 1 0 For Research Use Only Not for use in diagnostic procedures technologies Information in this document is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF NOTICE TO PURCHASER LIMITED USE LABEL LICENSE Research Use Only The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is n
19. ach v v Solution containing AmpliTaq Fast DNA TaqMan Fast Advanced Polymerase Uracil N glycosylase UNG Master Mix dNTPs with dUTP ROX dye passive pean d reference and optimized buffer components TM A TaqMan hPSC TaqMan hPSC Scorecard Panel Quick Jak Y T Scorecard Panel QRC Reference Card TaqMan Fast Advanced Master Mix Cat no 4444556 is also available separately from Life Technologies see page 38 Shipping and The TaqMan hPSC Scorecard Panels and Kits are shipped on wet ice Upon storage receipt transfer the entire shipment to 2 8 C for immediate storage Store the individual components as described below The performance of the products is guaranteed for 6 months from the date of purchase if stored and handled properly Component Storage and Handling TaqMan hPSC Scorecard Panel 384w or 96w FAST plates Store at 4 C 30 C Maintain in foil bag until ready to for use Briefly spin plates at 400 x 2 for 2 minutes prior to use MicroAmp Optical Adhesive Film Store at 4C 30 C Protect from dust TaqMan Gene Expression Master Mix Store at 2 C 8 C DO NOT FREEZE TaqMan Fast Advanced Master Mix Store at 22C 8 C DO NOT FREEZE Description of the System TaqMan hPSC Scorecard Panels How hPSC Scorecard Panels work Types of hPSC Scorecard Panels TaqMan hPSC Scorecard Panel 384w and TaqMan hPSC Scorecard
20. additional safety guidelines consult the SDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Chemical Waste Safety Chemical Waste Hazard Chemical Waste Safety Guidelines Waste Disposal CAUTION HAZARDOUS WASTE Refer to Safety Data Sheets SDSs and local regulations for handling and disposal To minimize the hazards of chemical waste Read and understand the Safety Data Sheets SDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consu
21. an 8 is ideal for downstream applications 25 cDNA Preparation Reverse Transcription of Total RNA Introduction This section provides instructions on generating single stranded cDNA from the total RNA by reverse transcription RT using the High capacity cDNA Reverse Transcription kit with RNase Inhibitor Materials needed e High capacity cDNA Reverse Transcription Kit with RNase Inhibitor Cat no 4374966 Perform RT 1 Allow the components of the High capacity cDNA Reverse Transcription Kit reaction with RNase Inhibitor to thaw on ice 2 Prepare 2X RT master mix by mixing the following components 1 sample 4 samples Component Per well 8 wells 8 wells sample 10X TaqMan RT Buffer 5 pL 50 pL 190 pL 25X dNTP Mix 2 pL 20 pL 76 uL 10X Random Primers 5 uL 50 pL 190 pL MultiScribe Reverse Transcriptase 50 U nL 2 5 pL 25 uL 95 uL RNase Inhibitor 20 U pL 2 5 pL 25 pL 95 pL RNase free water 8 uL 80 pL 304 uL TOTAL 25 pL 250 pL 950 pL 3 Place the 2X RT master mix on ice and mix gently Prepare RNA samples by diluting 1 ug total RNA in a total of 225 uL of RNase free water Add 225 uL of 2X RT master mix to the diluted RNA and mix well Aliquot 50 uL of the above RNA plus RT mix in 8 vertical wells of a 96 well plate or an 8 strip PCR tube see image below 8 9 10 11 12 _ _ Sample 1 Sample2 Sample3 Sample4 Continued on next page 26 Reverse Transcription
22. centration When the edges of the colonies are starting to pull away from the vessel carefully aspirate the Collagenase IV solution from the vessel without disturbing the attached cell layer and add 3 mL of ESC medium Dislodge the cells with a 5 mL pipet by gently blowing the cells off the surface of the vessel while pipetting up and down This will not only dislodge the colonies but also manually break them up into small fragments to be passaged Do not create a single cell suspension usually 5 10 pipetting motions will suffice to dislodge and resuspend the cells Transfer the cell clumps into a 15 mL tube Use an additional 2 mL of medium to collect any cell clumps remaining in the vessel and add to the 15 mL conical tube Allow the cells to gravity sediment for approximately 5 minutes This will permit the larger clumps to pellet out while allowing the removal of smaller clumps single cells and any iMEFs from the feeder layer that were dislodged through the process and still floating in the supernatant Aspirate the supernatant and then gently tap the tube to loosen the cell pellet from the bottom of the tube Add the appropriate amount of ESC medium for your passaging split ratio At this point you can continue to gently dissociate the cells into small clusters 50 500 cells by gentle pipetting Avoid making single cell suspensions Seed the cell clusters onto Geltrex matrix coated culture vessel containing MEF conditioned
23. culture vessels in several quick back and forth and side to side motions to disperse the cells across the surface of the vessels 7 Incubate the cells in a 37 C incubator with a humidified atmosphere of 5 CO 8 Use the MEF culture vessels within 3 4 days after plating Approximate Volume of MEF Volume of MEF Vessel size growth area medium Number of MEFs suspension 24 well plate 2 cm well 0 5 mL 5 0 x 10 well 20 uL 12 well plate 4 cm well 1 mL 1 0 x 10 well 40 uL 6 well plate 10 cm well 2mL 2 5 x 10 well 0 1 mL 60 mm dish 20 cm 5mL 5 0 x 10 0 2 mL 100 mm dish 60 cm 10 mL 1 5 x 10 0 6 mL 25 cm flask 25 cm 5mL 6 3 x 10 0 25 mL 75 cm flask 75 cm 15 mL 1 9 x 106 0 75 mL 36 Appendix C Background Information TaqMan Chemistry TaqMa n probes TaqMan probes are dual labeled hydrolysis probes that increase the specificity of real time PCR assays TaqMan probes contain e A reporter dye for example FAM dye linked to the 5 end of the probe e Anon fluorescent quencher NFQ at the 3 end of the probe e MGB moiety attached to the NFQ TaqMan MGB probes also contain a minor groove binder MGB at the 3 end of the probe MGBs increase the melting temperature Tm without increasing probe length allowing for the design of shorter probes How TaqMan real 1 An oligonucleotide probe is constructed with a fluorescent reporter dye time chemistry bound to the 5 end and a
24. e Technologies only under a separate agreement For information on obtaining additional rights please contact outlicensing dlifetech com or Out Licensing Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation and or its affiliate s or their respective owners Parafilm is a registered trademark of Bemis Company Inc TaqMan is a registered trademark of Roche Molecular Systems Inc used under permission and license 2013 Life Technologies Corporation All rights reserved Table of Contents PRG eC ee TE A 2 About This Guide 32 dece OTRO ee ate ree pete dre etede eel eti a esee isset Eia 2 ignit raainiomium eR 3 Kit Contents and Storage vescovo S eee ertet eee eet 3 Deseriptionof the 5ystem asse ckaec dau sten ete ee etd ee ete edes 5 TaqMan hPSC Scorecard Panel Workflow 2 tela cii bete bri iucunda i beet inb 7 Sample Generation me anii 8 Undifferentiated Cells Feeder Dependent Culture sse n 8 Undifferentiated Cells Feeder Free Culture in StemPro RESC SFM ccccsccssessesessesseecssesseeessesnceseseaneeeese 11 Undifferentiated Cells Feeder Free Culture in Essential 8 Medium sse 14 Randomly Differentiated Cells Embryoid Bodies sse eee 17 Sample Preparation i coins oi oc ot aaa betae tod vec tinte ta vaca e Re
25. e plates and supplies The following media reagents and culture vessels are needed for culturing and harvesting undifferentiated ESCs or iPSCs maintained as feeder free cultures on in complete Essential 8 medium For instructions on preparing the media and reagents listed below see Appendix A Recipes page 28 For instructions on coating culture vessels with Geltrex matrix see Appendix B Preparing Culture Vessels page 34 e Essential 8 Medium e 10X Collagenase IV Solution 10 mg mL e Geltrex matrix coated culture vessels 1 Coatthe culture vessels with Geltrex matrix at least 1 hour before passaging the cells Warm the appropriate amount of 10 mg mL 10X Collagenase IV solution and DMEM F 12 to 37 C in a water bath 2 Warm complete Essential 8 medium required for that day at room temperature until it is no longer cool to the touch approximately 20 30 minutes Do not warm the Essential 8 medium at 37 C 3 When the ESC or iPSC culture has reached the desired confluency to yield at least 5 x 10 cells per sample aspirate off the cell culture medium i e Essential 8 medium 4 Gently add 10 mg mL Collagenase Type IV 10X solution to the culture vessel containing ESCs or iPSCs Adjust the volume of Collagenase IV for various dish sizes refer to Table 3 page 16 Continued on next page Undifferentiated Cells Feeder Free Culture in Essential 8 Medium continued Culture and harvest 5 Incubate for 3 5
26. e recommend treating the isolated total RNA with the DNA free Kit which digests the contaminating DNA to levels below the limit of detection by routine PCR e DNA free Kit Cat no AM1906 contains rDNase I 10X DNase I Buffer DNase Inactivation Reagent and nuclease free water e We recommend conducting the reactions in 0 5 mL tubes to facilitate removal of the supernatant after treatment with the DNase Inactivation Reagent e DNA free reactions can be conducted in 96 well plates We recommend using V bottom plates because their shape makes it easier to remove the RNA from the pelleted DNase Inactivation Reagent at the end of the procedure e The recommended reaction size is 10 100 uL A typical reaction is 50 pL e Routine DNase treatment removes 2 ug of genomic DNA from 50 uL reaction with lt 200 pg mL nucleic acid refer to product insert if more rigorous DNase treatment is needed 1 Fora 50 uL reaction combine the following reagents in a clean DNase and RNase free 0 5 mL microcentrifuge tube and mix gently RNA Sample 1 10 ug 10X DNase I Reaction Buffer 5 pL rDNase I 2 Units 1 uL DEPC treated water to bring reaction to 50 uL X uL Total 50 pL Incubate the tube at 37 C for 20 30 minutes Resuspend the DNase Inactivation Reagent by flicking or vortexing the tube add 5 uL 0 1 volume of the resuspended inactivation reagent to the reaction mix and mix well Incubate for 2 minutes at room temperature mixing t
27. ed culture vessels This section provides instructions on harvesting undifferentiated ESCs or iPSCs that are maintained as feeder dependent cultures on inactived murine embryonic fibroblast MEF feeder cells for total RNA extraction You will need to harvest at least 5 x 10 cells per sample to isolate sufficient total RNA for the subsequent steps of the workflow IMPORTANT Feeder dependent ESCs or iPSCs should be cultured feeder free on Geltrex matrix coated culture vessels for one passage in MEF conditioned medium before the cells are harvested and total RNA is isolated e DMEM F 12 with GlutaMAX I Cat no 10565 018 e KnockOut Serum Replacement KSR Cat no 10828 010 e MEM Non Essential Amino Acids Solution 10 mM Cat no 11140 050 e p Mercaptoethanol 1000X liquid Cat no 21985 023 e Basic Fibroblast Growth Factor bFGF recombinant human Cat no PGH0264 e Collagenase Type IV Cat no 17104 019 e Attachment Factor Cat no S 006 100 e DMEM with GlutaMAX I high glucose Cat no 10569 010 e Fetal Bovine Serum ESC Qualified Cat no 16141 079 e DPBS no Calcium no Magnesium Cat no 14190 144 e Geltrex hESC qualified Cat no A1413302 e TRIzol Reagent Cat no 18596 018 e Gibco Mouse Embryonic Fibroblasts Irradiated Cat no 1520 100 e Cell Scraper Falcon Cat no 353085 e Appropriate tissue culture plates and supplies The following media reagents and culture
28. ell or the 96 well plate using fresh tips each time as shown below For 96 well plates one well is sufficient to load one row of the plate cDNA strip TaqMan hPSC Scorecard 384w plate R 140 pL 999933 12x 10L 989883939833 999923 89233 00000 O00000 OO00 000000 000000 O00000 O00000 000000 000000 000000 O00000 000000 O00 000000 000000 000000 Q00000 0000 O000 O0000 200000 O00000 O00000 000000 000000 999999 900099 000999099009 9909990 e 00090 00000 0 06 000000 000000 e O00000 O00000 O OOOO0O0 000000 O00000 O00000 000000 000000 000000 e O00000 600000 00000 O0000 000000 O 909000 899999 990000 900009 900909 000000 O00000 000000 000000 000000 Sample 1 Sample 1 Sample 2 Sample 3 Sample 4 4 cDNA samples per 384 well plate cDNA strip TaqMan hPSC Scorecard 96w FAST plate 140 uL 000000000000 12 10 L OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO 1 cDNA sample per 96 well plate Du o0000000 Continued on next page qRT PCR Using the TaqMan hPSC Scorecard Panel continued Run the qRT PCR continued 4 Seal the plate with the MicroAmp Optical Adhesive Film and centrifuge it at 600 x g for 2 minutes Place the plate in a compatible RT PCR instrument equipped with the appropriate thermal block IMPORTANT TaqMan hPSC Scorecard Panel 96w FAST must be run on RT PCR sys
29. ell plate 10 cm well 2 0 mL per well 12 well plate 4 cm well 1 0 mL per well 24 well plate 2 cm well 0 5 mL per well 35 mm dish 10 cm 2 0 mL 60 mm dish 20 cm 5 0 mL 100 mm dish 60 cm 12 0 mL Continued on next page Randomly Differentiated Cells Embryoid Bodies continued Harvest cells Option 1 7 day EB suspension Harvest cells Option 2 4 day EB suspension and adherent culture On day 7 of EB suspension gently transfer the cells and the medium from the Petri dish into a 15 mL conical tube Use an additional 5 mL of DPBS to collect any remaining EBs from the culture dish and add into the conical tube Allow the EBs to sediment down by gravity for 10 15 minutes and then aspirate off the supernatant i e spent EB medium Using a P1000 pipettor add 1 mL of TRIzol reagent and pipette up and down to assist in properly breaking up the cell clumps Incubate the EBs for 2 3 minutes Repeat pipetting and incubation if the EBs require more time to be lysed Collect the slurry into a sterile RNAse free microcentrifuge tube Store at 80 C until ready for RNA isolation On Day 4 gently transfer the cells and the medium from the Petri dish into a 15 mL conical tube Use an additional 5 mL of DPBS to collect any remaining EBs from the culture dish and add into the conical tube Allow the EBs to sediment down by gravity for 10 15 minutes in the cell culture hood Aspirate the supernatan
30. ere we cover suspension EB culture which is recommended for a maximum of 7 days If extended EB culture is desired we recommend seeding the EBs on a Geltrex matrix coated culture vessel and continuing as an adherent culture as described in Harvest cells Option 2 4 day EB suspension and adherent culture page 19 until total RNA isolation e DMEM F 12 with GlutaMAX I Cat no 10565 018 e KnockOut Serum Replacement KSR Cat no 10828 010 e MEM Non Essential Amino Acids Solution 10 mM Cat no 11140 050 e p Mercaptoethanol 1000X liquid Cat no 21985 023 e DPBS no Calcium no Magnesium Cat no 14190 144 e TRIzol Reagent Cat no 18596 018 e Cell Scraper Falcon Cat no 353085 e 60 mm Petri dish non tissue culture treated or ultra low binding dish e Appropriate tissue culture plates and supplies e Geltrex hESC qualified Cat no A1413302 for optional adherent EB culture The following media and matrix coated culture vessels are needed for creating and harvesting EBs for total RNA isolation For instructions on preparing the media listed below see Appendix A Recipes page 28 For instructions on coating culture vessels with Geltrex matrix see Appendix B Preparing Culture Vessels page 34 e EB medium e Geltrex matrix coated culture vessels for optional adherent EB culture Embryoid Bodies EBs are generated at a normally scheduled passage by plating ESCs or iPSCs into n
31. ganic phase extraction using the TRIzol reagent Note that column based purification methods see page 23 may also yield high quality RNA e TRIzol reagent Cat no 15596 026 e Chloroform Sigma Cat no C 2432 e Isopropanol Sigma Cat no 19516 e Ethanol Sigma Cat no E7023 e UltraPure DNase RNase Free Distilled Water Cat no 10977 CAUTION TRIzol Reagent contains phenol toxic and corrosive and guanidine isothiocyanate an irritant and may be a health hazard if not handled properly Always work with TRIzol Reagent in a fume hood and always wear a lab coat gloves and safety glasses For more information refer to the TRIzol Reagent SDS Safety Data Sheet available from our website at www lifetechnologies com support 1 Incubate the lysate with TRIzol reagent from the last step of the harvesting procedure at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes To the cells in TRIzol reagent add 0 2 mL of chloroform per 1 mL of TRIzol reagent and shake the tube vigorously by hand for 15 seconds 3 Incubate the sample at room temperature for 2 3 minutes and centrifuge at 12 000 x 2 for 15 minutes at 4 C N Note The mixture separates into a lower red phenol chloroform phase an interphase and a colorless upper aqueous phase RNA remains exclusively in the aqueous phase The upper aqueous phase is 50 of the total volume 4 Carefully
32. he reaction occasionally Centrifuge at 10 000 x g for 1 5 minutes and transfer the RNA to a fresh tube RNA is now ready for reverse transcription Total RNA Isolation Using the TRIzol Plus RNA Purification Kit Alternative PureLink Column Based Method Introduction The TRIzol Plus RNA Purification Kit provides a simple reliable and rapid method for isolating high quality total RNA from a wide variety of samples including animal and plant cells and tissue bacteria and yeast The kit utilizes the strong lysis capability of TRIzol reagent followed by a convenient and time saving silica cartridge purification protocol from the PureLink RNA Mini Kit to purify total RNA Materials needed e TRIzol Plus RNA Purification kit Cat no 12183 555 e Chloroform Sigma Cat no C 2432 e Ethanol Sigma Cat no E7023 e UltraPure DNase RNase Free Distilled Water Cat no 10977 Isolate total RNA 1 Incubate the lysate with TRIzol reagent from the last step of the harvesting using the TRIzol procedure at room temperature for 5 minutes to allow complete dissociation Plus RNA of nucleoprotein complexes 2 To the cells in TRIzol reagent add 0 2 mL of chloroform per 1 mL of TRIzol reagent and shake the tube vigorously by hand for 15 seconds Purification Kit 3 Incubate the sample at room temperature for 2 3 minutes and centrifuge at 12 000 x 2 for 15 minutes at 4 C 4 Transfer 600 uL of the colorless upper pha
33. in Cartridge into the same Collection Tube Repeat Steps 5 6 once Centrifuge the Spin Cartridge and Collection Tube at 12 000 x g for 1 minute at room temperature to dry the membrane with attached RNA Discard the Collection Tube and insert the Spin Cartridge into a Recovery Tube Add 30 100 uL RNase Free Water to the center of the Spin Cartridge Incubate at room temperature for 1 minute Centrifuge the Spin Cartridge with the Recovery Tube for 2 minutes at 2 12 000 x g at room temperature The recovery tube contains the purified total RNA RNA Quantification and Quality Introduction Asses total RNA amount and quality We recommend using total RNA that is Between 0 002 and 0 2 ug uL Less than 0 005 of genomic DNA by weight Dissolved in a PCR compatible buffer Free of RNase activity Free of inhibitors of reverse transcription and PCR Nondenatured n IMPORTANT Denaturation of the RNA is not necessary and may reduce the yield of cDNA for some gene targets Use NanoDrop to quantify extracted RNA sample Quality of RNA is best assessed using A 9 289 with the recommended value close to 2 0 RNA integrity can be further assessed by running the samples on a 1 Agarose gel and assessing the 2 1 ratio of the 28s and 18s RNA bands and the absence of degraded RNA that appears as small molecular weight smear If using Bioanalyzer a RIN RNA integrity number value of higher than 5 maybe sufficient but higher th
34. ion 5 When the edges of the colonies are starting to pull away from the vessel carefully aspirate the Collagenase IV solution from the vessel without disturbing the attached cell layer and gently rinse the cells with 3 mL of pre warmed StemPro wash solution 6 Aspirate the wash solution and add the appropriate volume of pre warmed complete StemPro hESC SFM medium refer to Table 2 page 13 7 Use a sterile cell scraper to gently remove the cell clumps Pipette the cell suspension across the plate surface with a 5 mL pipette to break up the detached colonies into smaller clumps Do not create a single cell suspension usually 3 5 pipetting motions will suffice to dislodge and resuspend the cells 8 Gently transfer the cell clumps into a 15 mL tube Depending on the culture vessel use an additional 1 3 mL of pre warmed complete StemPro hESC SFM medium to collect any cell clumps remaining in the vessel and add to the 15 mL conical tube 9 Centrifuge cell suspension at 200 x g for 2 minutes or allow the cells to gravity sediment at room temperature for 5 10 minutes 10 Aspirate off the Geltrex solution from the fresh Geltrex matrix coated vessel and add the appropriate volume of pre warmed complete StemPro hESC SFM medium refer to Table 2 page 13 11 Following centrifugation or gravity sedimentation of the cell suspension step 9 aspirate off the supernatant and gently re suspend the cell clumps with the appropria
35. ions handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches Safety Data Sheets SDSs are available at www lifetechnologies com sds The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support 43 Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit lifetechnologies com support or email techsupport dlifetech com technologies lifetechnologies com 13 June 2013
36. lt the SDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Handle chemical wastes in a fume hood After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations n IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply 41 Biological Hazard Safety 42 WARNING BIOHAZARD Biological samples such as tissues body fluids and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective eyewear clothing and gloves Read and follow the guidelines in these publications
37. mL of complete StemPro hESC medium aseptically mix the following components StemPro hESC medium without bFGF can be stored at 2 8 C for up to 2 weeks Component Volume DMEM F 12 with HEPES 90 8 mL StemPro hESC Supplement 2 0 mL BSA 25 7 2 mL B Mercaptoethanol 55 mM 182 pL Basic FGF 10 ug mL 80 pL Prepare the StemPro hESC medium without bFGF and then supplement with fresh bFGF to a final concentration of 8 ng mL when the medium is used 1 To prepare 100 mL of StemPro wash solution aseptically mix the following components DMEM F 12 with HEPES 100 mL BSA 25 0 2 mL Filter sterilize the solution through a 0 22 um filter and store at 2 8 C for up to 2 weeks Thaw the frozen Essential 8 Supplement at 2 8 C overnight Do not thaw the frozen supplement at 37 C Mix the thawed supplement by gently inverting the vial a couple of times remove 10 mL from the bottle of DMEM F 12 HAM 1 1 and then aseptically transfer the entire contents of the Essential 8 Supplement to the bottle of DMEM F 12 HAM 1 1 Swirl the bottle to mix and to obtain 500 mL of homogenous complete medium Complete Essential 8 medium can be stored at 2 8 C for up to 2 weeks Before use warm complete medium required for that day at room temperature until it is no longer cool to the touch Do not warm the medium at 37 C To prepare 100 mL of complete EB medium aseptically mix the components listed below Comple
38. nee Aiai raada 20 Total RNA Isolation by TRIzol Organic Phase Extraction Recommended Method 20 Optional DNase Treatments 5 mer ete dee oe n tegere p eee eee es 22 Total RNA Isolation Using the TRIzol Plus RNA Purification Kit Alternative PureLink Column Based Method 5 ed Aaa eere reete pee ee ene e deer eere er iecit 23 RNA Quantificationrand Quality rete eee te e tt te tete ertet 25 cDNA PreparalloD scu cte o en rr Puedo pulo Fa ntt ke Dar ctun YE Eee EO cue Qe 26 Reverse Transcription of Total RNA notieren er rt ped dete eb eoe dna ete SaS 26 TedMall Ts P OR coast hice Dau or odi rue e REN REC mu Na e Nae eR a Cd 28 qRT PCR Using the TaqMan hPSC Scorecard Panel ie SE Eh dotati editis 28 AppehdbcA Recipes iso cuoc e aaea iaai aa QUEM edd sue E NQUODE 31 Medlia arid Redgents x25 euet dn e eee ete tinet eeu 31 Appendix B Preparing Culture Vessels essen nennen 34 Coating Culture Vessels with Geltrex Matrix deeteientbi enkettie onset eati ono e dina 34 MEF Culture Dishes tee tt ie D teet dinde dee e iei a bed E eden 35 Appendix C Background Information cccccccceeeeeeeeseeeeeeeeeeeeeeaaaaaeeeeeeeeeseeaeaeaeeeeeeeeeeenaaa 37 TagNLun OHeBUSHU ede aedi aes nea tet adr ge relati ANS aa Taeao ie ieia tabe de ide ete 37 Appendix D Ordering Information eeeseeeesssesssessseeeeeeeeeee nennen nennen nennen 38 PLOGUCHS P p 38
39. not need to rinse off the Geltrex matrix solution from the culture dish after removal Cells can now be passaged directly onto the Geltrex matrix coated culture dish Culture vessel Surface area Volume of Geltrex matrix dilution 6 well plate 10 cm well 15 mL well 12 well plate 4 cm well 750 uL well 24 well plate 2 cm well 350 uL well 35 mm dish 10 cm 15mL 60 mm dish 20 cm 3 0 mL 100 mm dish 60 cm 6 0 mL MEF Culture Dishes Gelatin coating culture vessels Thawing MEFs 1 Cover the whole surface of each culture vessel with Attachment Factor AF solution and incubate the vessels for 30 minutes at 37 C or for 2 hours at room temperature Note AF Cat no S 006 100 is a sterile 1X solution containing 0 1 gelatin available from Life Technologies see page 39 for ordering information Using sterile technique in a laminar flow culture hood completely remove the AF solution from the culture vessel by aspiration Note It is not necessary to wash the culture surface before adding cells or medium Coated vessels may be used immediately or wrapped in Parafilm sealing film and stored at room temperature for up to 24 hours Remove the cryovial containing inactivated MEFs from the liquid nitrogen storage tank Briefly roll the vial between hands to remove frost and swirl it gently in a 37 C water bath When only a small ice crystal remains in the vial remove it from water bath Spray
40. nued Analyze the results 30 Analyze the gene expression data from the TaqMan hPSC Scorecard Panels using the web based hPSC Scorecard Analysis Software available at www lifetechnologies com scorecarddata The hPSC Scorecard Analysis Software summarizes all key experimental results including pluripotency and differentiation potential on a single dashboard It also allows you to tag and filter experiments view expression correlation and box plots and export experimental results and data as a PDF or as a spreadsheet Appendix A Recipes Media and Reagents Basic FGF stock 1 To prepare 10 mL of 10 ug mL Basic FGF solution aseptically mix the solution following components Basic FGF 100 pg DPBS without Ca and Mg 9 8 mL 10 BSA 100 uL 2 Aliquot and store the Basic FGF solution at 20 C for up to 6 months 0 5 mM EDTA in 1 To prepare 50 mL of 0 5 mM EDTA in DPBS aseptically mix the following DPBS components in a 50 mL conical tube DPBS without Ca and Mg 50 mL 0 5 M EDTA 50 uL 2 Filter sterilize the solution through a 0 22 um filter and store at room temperature for up to 6 months Collagenase Type IV 10X Collagenase Type IV solution 10 mg mL for 50 mL solution 1 Add 50 mL of DMEM F 12 to 500 mg of Collagenase Type IV to make a 10 mg mL stock solution 10X 2 Gently vortex to suspend and filter sterilize the solution through a 0 22 um filter This solution can be stored at 2 8 C for up to 2
41. of Total RNA continued Perform RT 7 Run the RT reaction in a thermal cycler using conditions as listed below reaction continued Step Temperature Time 1 25 C 10 minutes 2 37 C 120 minutes 3 85 C 5 minutes 4 4 C hold 8 Proceed to TaqMan qRT PCR page xx If you do not proceed immediately to PCR amplification store all cDNA samples at 15 C to 25 C To minimize freeze thaw cycles store the cDNA in smaller aliquots 27 TaqMan qRT PCR qRT PCR Using the TaqMan hPSC Scorecard Panel Introduction Materials needed Run the qRT PCR 28 This section provides instructions on analyzing your cDNA samples by qRT PCR using the TaqMan hPSC Scorecard Panel e TaqMan hPSC Scorecard Panel TaqMan hPSC Scorecard Panel 384w or TaqMan hPSC Scorecard Panel 96w FAST e TaqMan Fast Advanced Master Mix 96 well format for running in FAST mode using the TaqMan hPSC Scorecard Panel 96w FAST e TaqMan Gene Expression Master Mix 384 well format using the TaqMan hPSC Scorecard Panel 384w e MicroAmp Optical Adhesive Film 1 Dilute each well containing 50 pL cDNA with 20 uL PCR water for a final volume of 70 uL 2 Add 70 pL 2X TaqMan Gene Expression Master Mix if using the TaqMan hPSC Scorecard Panel 384w or 70 uL 2X TaqMan Fast Advanced Master Mix if using the TaqMan hPSC Scorecard Panel 96w FAST 3 Load 10 uL per well using multichannel pipette onto the 384 w
42. ombine the appropriate TaqMan master mix with your cDNA sample and RNase free water and reconstitute each well of the TaqMan hPSC Scorecard Panel by adding 10 uL of the reaction mixture per well e Load and run the plates on a compatible real time PCR RT PCR instrument using either standard or Fast thermal cycling conditions e Analyze the gene expression data using the web based hPSC Scorecard Analysis Software to confirm the pluripotency of the samples and predict their differentiation potential and outcome The hPSC Scorecard Analysis Software is available at www lifetechnologies com scorecarddata TaqMan hPSC Scorecard Panels are available as 384 well plates Cat nos A15870 A15872 or as 96 well FAST plates Cat nos A15876 A15871 for use with Fast thermal cycling conditions e TaqMan hPSC Scorecard Panel 384w are 384 well MicroAmp optical assay plates which allow the analysis of four separate cDNA samples under standard thermal cycling conditions e TaqMan hPSC Scorecard Panel 96w FAST are 96 well MicroAmp optical Fast thermal cycling plates which reduce quantitative PCR run times to less than 40 minutes when used under Fast thermal cycling conditions in a compatible RT PCR system Each 96 well plate allows the analysis of one cDNA sample Continued on next page Description of the System continued Compatible TaqMan Each well in a TaqMan hPSC Scorecard Panel contains a pair of unlabeled PCR Mas
43. on tissue culture treated dishes to prevent attachment and allowing them to aggregate to form EBs 1 Culture ESCs or iPSCs on MEF feeder cells or in the desired feeder free condition StemPro hESC SFM MEF CM or Essential 8 Medium until the cells are approximately 90 confluent 2 Aspirate off the culture medium from the culture plates or dishes and then add 1 mL pre warmed EB medium to each well of 6 well plate or to each 35 mm dish 2 mL to each 60 mm dish or 3 mL to each 100 mm dish Continued on next page 17 Randomly Differentiated Cells Embryoid Bodies continued Generate EBs from ESCs iPSCs continued 18 3 10 Roll the StemPro EZPassage disposable stem cell passaging tool across the entire dish or plate in one direction left to right Rotate the culture dish or plate 90 degree and roll the StemPro EZPassage disposable stem cell passaging tool across the entire dish or plate Use a cell scraper to gently detach the cells off the surface of the culture vessel Gently transfer the cell clumps using a 5 mL pipette into a 15 mL conical tube Do not break the cell clumps into small pieces Depending on the culture vessel use an additional 1 3 mL of pre warmed EB medium see step 2 page 17 to collect any cell clumps remaining in the vessel and add to the 15 mL conical tube Transfer the cell clumps to a 60 mm Petri dish non tissue culture treated or ultra low binding dish in a total of 5 mL of E
44. or 30 minutes at 37 C or for 1 hour at room temperature For MEF CM generation a T 175 flask is recommended Note AF Cat no S 006 100 is a sterile 1X solution containing 0 1 gelatin available from Life Technologies see page 39 for ordering information 2 Using sterile technique in a laminar flow culture hood completely remove the AF solution from the culture vessel by aspiration just prior to use Coated vessels may be used immediately or stored at room temperature for up to 24 hours Note It is not necessary to wash the culture surface before adding cells or medium 3 Plate 9 4 x 10 Mitomycin C treated or irradiated MEFs in a 1 175 flask coated with AF and containing 45 mL of MEF medium Allow the cells to attach overnight in the incubator under normal growth conditions The following day replace the MEF medium with 90 mL of ESC medium 5 Collect the ESC medium now considered MEF CM from the flasks after 24 hours of conditioning This method of producing MEF CM can be repeated up to seven days in a row 6 Each day filter sterilize the collected MEF CM through a 0 22 uM filter Filtered MEF CM can be used immediately or stored at 20 C until use 7 Atthe time of use supplement the MEF CM with fresh bFGF at a final concentration of 4 ng mL Continued on next page Media and Reagents continued StemPro hESC medium StemPro wash solution Essential 8 medium Embryoid Body EB medium To prepare 100
45. ot for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing dlifetech com or Out Licensing Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 NOTICE TO PURCHASER LIMITED LICENSE Diagnostic uses of this product require a separate license from Roche For information on obtaining additional rights please contact outlicensing alifetech com or Out Licensing Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 NOTICE TO PURCHASER LIMITED LICENSE Patents covering oligonucleotide conjugates of Minor Groove Binder MGB are owned by ELltech Group and licensed to Life Technologies for use in assays whereby the detection is mediated by a 5 nuclease activity of a polymerase enzyme the 5 Nuclease Process The purchase of this product includes a license to use only this amount of product solely for the purchaser s own use and may not be used for any other commercial use including without limitation human in vitro diagnostics repackaging or resale in any form including resale by purchasers who are licensed to make and sell kits for use in the 5 Nuclease Process Corresponding products conveying commercial and diagnostic use rights for MGB may be obtained from Lif
46. quencher on the 3 end works While the probe is intact the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer through space 2 If the target sequence is present the probe anneals between primer sites and is cleaved by the 5 nuclease activity of the Taq DNA polymerase during extension This cleavage of the probe e Separates the reporter dye from the quencher increasing the reporter dye signal e Removes the probe from the target strand allowing primer extension to continue to the end of the template strand Thus inclusion of the probe does not inhibit the overall PCR process 3 Additional reporter dye molecules are cleaved from their respective probes with each cycle resulting in an increase in fluorescence intensity proportional to the amount of amplicon produced The higher the starting copy number of the nucleic acid target the sooner a significant increase in fluorescence is observed 37 Appendix D Ordering Information Products TaqMan hPSC Various components of the TagMan hPSC Scorecard Panels are also available Scorecard Panel separately from Life Technologies For more information about the following products products refer to our website at www lifetechnologies com or contact Technical Support page 43 Product Quantity Catalog no TaqMan hPSC Scorecard Panel 384w 1 plate A15870 TaqMan hPSC Scorecard
47. re 80 90 confluent changing the medium daily 17 On the day of harvesting aspirate the culture medium and wash the cells once with 5 mL of DPBS for 2 3 minutes 18 Aspirate the DPBS and discard 19 Add 1 mL of TRIzol reagent and incubate for 2 3 minutes Scrape the plate with a sterile cell scraper and collect the slurry into a sterile RNAse free microcentrifuge tube 20 Store the cells at 80 C until ready for RNA isolation Culture and harvest 16 cells continued Table 3 Volume of Collagenase IV 10 mg mL and complete Essential 8 medium required Volume of Volume of complete Culture Vessel Surface Area Collagenase IV Essential 8 medium 6 well plate 10 cm well 1 0 mL per well 2 0 mL per well 12 well plate 4 cm well 0 5 mL per well 1 0 mL per well 24 well plate 2 cm well 0 25 mL per well 0 5 mL per well 35 mm dish 10 cm 1 0 mL 2 0 mL 60 mm dish 20 cm 2 0 mL 4 0 mL 100 mm dish 60 cm 6 0 mL 10 0 mL 16 Randomly Differentiated Cells Embryoid Bodies Introduction Materials needed Prepare media and matrix coated culture vessels Generate EBs from ESCs iPSCs This section provides instructions on generating embryoid bodies EBs for random differentiation from ESCs or iPSCs maintained as feeder dependent or feeder free cultures and their subsequent harvest for total RNA extraction IMPORTANT There are several methods for creating EBs for random differentiation H
48. remove the upper aqueous phase and transfer to a new tube Avoid drawing any of the interphase or organic layer into the pipette when removing the aqueous phase Continued on next page Total RNA Isolation by TRIzol Organic Phase Extraction continued Isolate total RNA by 5 TRIzol organic phase extraction continued 6 10 Add 0 5 mL of 100 isopropanol to the aqueous phase per 1 mL of TRIzol reagent and incubate at room temperature for 10 minutes Centrifuge at 12 000 x g for 10 minutes at 4 C Carefully remove the supernatant from the RNA pellet and wash with 1 ml 75 ethanol w gt Note The RNA is often invisible prior to centrifugation and forms a gel like pellet on the side and at the bottom of the tube upon centrifugation Centrifuge the tube at 7500 x g for 5 minutes at 4 C Discard the supernatant and air dry the RNA pellet for 5 10 minutes Resuspend the RNA pellet in 20 50 uL RNase free water 21 Optional DNase Treatment Introduction Materials needed Guidelines for using the DNA ree Kit DNA free Kit procedure 22 One key variable to the success of any RT PCR experiment is the quality of the template RNA DNA removal is critical for ensuring high quality RNA because DNA can serve as a template during the PCR portion of the experiment resulting in false positives background etc Ideally the total RNA sample should have less than 0 005 of genomic DNA by weight W
49. se containing the RNA to a fresh RNase free tube and add an equal volume of 100 ethanol to obtain a final ethanol concentration of 50 Mix well by vortexing 5 Invert the tube to disperse any visible precipitate that may form after adding ethanol Proceed to next step using the column 6 Transfer up to 700 pL of sample to a Spin Cartridge with a Collection Tube and centrifuge at 12 000 x 2 for 15 seconds at room temperature Discard the flow through and reinsert the Spin Cartridge into the same Collection Tube Repeat above two steps until the entire sample has been processed Optional If your downstream application requires DNA free total RNA proceed to On Column PureLink DNase Treatment during RNA Purification at this time for details see the PureLink RNA Mini Kit user guide available from www lifetechnologies com 9 Add 700 uL Wash Buffer I to the Spin Cartridge Centrifuge at 12 000 x g for 15 seconds at room temperature 10 Discard the flow through and the Collection Tube Insert the Spin Cartridge into a new Collection Tube Continued on next page 23 Total RNA Isolation Using the TRIzol Plus RNA Purification Kit continued Isolate total RNA 11 using the TRIzol 12 Plus RNA Purification Kit 13 continued 14 15 16 24 Add 500 pL Wash Buffer II with ethanol to the Spin Cartridge Centrifuge at 12 000 x g for 15 seconds at room temperature Discard the flow through and reinsert the Sp
50. sion at 200 x g for 2 minutes or allow the cells to gravity sediment at room temperature for 5 10 minutes 11 Aspirate off the Geltrex solution from the fresh Geltrex matrix coated vessel and add the appropriate volume of pre warmed complete Essential 8 medium refer to Table 3 page 16 12 Following centrifugation or gravity sedimentation of the cell suspension step 10 aspirate off the supernatant and gently re suspend the cell clumps with the appropriate amount of complete Essential 8 medium 13 Add the desired amount of cell suspension into each new Geltrex matrix coated vessel according to the desired split ratio w gt Note The split ratio is variable though generally between 1 4 and 1 6 If the cells are overly dense and crowding increase the ratio and if the cells are sparse decrease the ratio Cells will need to be split every 4 6 days based upon their appearance 14 Move the culture vessel in several quick short back and forth and side to side motions to disperse the cells evenly across its surface and then return the vessel to the incubator 15 The next day gently aspirate the medium to remove the non attached cells and replace it with fresh complete Essential 8 medium Replace the spent medium with fresh medium every day thereafter Continued on next page 15 Undifferentiated Cells Feeder Free Culture in Essential 8 Medium continued Continue to culture the cells until they a
51. t i e spent EB medium and replace it with 5 mL of fresh EB medium Transfer the cells to a fresh 60 mm tissue culture treated dish coated with Geltrex matrix If using a different size culture dish refer to Table 4 for the volume EB of medium needed Place the dish containing the cells in the 37 C 5 CO incubator and change the spent medium every other day Allow the EBs to attach and the contents of the EBs to grow out from the EBs to obtain adherent cell types On Day 7 and 14 of total differentiation or as desired aspirate off the EB medium and wash the cells once with 5 mL of DPBS for 2 3 minutes Aspirate off the DPBS wash Add 1 mL of TRIzol reagent and incubate for 2 3 minutes Scrape the plate with a sterile cell scraper and collect the slurry into an RNAse free microcentrifuge tube Store at 80 C until ready for RNA isolation 19 Sample Preparation Total RNA Isolation by TRIzol Organic Phase Extraction Recommended Method Introduction Materials needed Isolate total RNA by TRIzol organic phase extraction 20 This section provides instructions on extracting total RNA from the ESCs and iPSCs by TRIzol organic phase extraction to use as a template for synthesis of single stranded cDNA You will need at least 5 x 10 cells harvested per sample to isolate sufficient total RNA for the reverse transcription reaction Note For optimal performance we recommend isolating total RNA by or
52. te EB medium can be stored at 2 8 C for up to 1 week Component Volume DMEM F 12 1X with GlutaMAX I 79 mL KnockOut Serum Replacement KSR 20 mL MEM Non essential Amino Acids Solution 10 mM 1mL B Mercaptoethanol 1000X 100 uL 33 Appendix B Preparing Culture Vessels Coating Culture Vessels with Geltrex Matrix Coating protocol 34 1 Thaw a 5 mL bottle of Geltrex LDEV Free hESC Qualified Reduced Growth Factor Basement Membrane Matrix at 2 8 C overnight Dilute the thawed Geltrex matrix solution 1 1 with cold sterile DMEM F 12 to prepare 1 mL aliquots in tubes chilled on ice These aliquots can be frozen at 20 C or used immediately Note The aliquot volumes of the 1 1 diluted Geltrex matrix solution may be adjusted according to your needs To create working stocks dilute an aliquot of Geltrex matrix solution 1 50 with cold DMEM on ice for a total dilution of 1 100 Note An optimal dilution of the Geltrex matrix solution may need to be determined for each cell line Try various dilutions from 1 30 to 1 100 Quickly cover the whole surface of each culture dish with the Geltrex matrix solution see table below Incubate the dishes in a 37 C 5 CO incubator for 1 hour Geltrex matrix coated culture dishes can now be used or stored at 2 8 C for up to a week Do not allow dishes to dry Aspirate the diluted Geltrex matrix solution from the culture dish and discard You do
53. te amount of complete StemPro hESC SFM medium 12 Add the desired amount of cell suspension into each new Geltrex matrix coated vessel according to the desired split ratio N Note The split ratio is variable though generally between 1 4 and 1 6 If the cells are overly dense and crowding increase the ratio and if the cells are sparse decrease the ratio Cells will need to be split every 4 6 days based upon their appearance 13 Move the culture vessel in several quick short back and forth and side to side motions to disperse the cells evenly across its surface and then return the vessel to the incubator 14 The next day gently aspirate the medium to remove the non attached cells and replace it with fresh complete StemPro hESC SFM medium Replace the spent medium with fresh medium every day thereafter Continued on next page 12 Undifferentiated Cells Feeder Free Culture in StemPro hESC SFM continued Culture and harvest 15 cells continued medium daily 16 On the day of harvesting aspirate the culture medium and wash the cells once with 5 mL of DPBS for 2 3 minutes 17 Aspirate the DPBS and discard Continue to culture the cells until they are 80 90 confluent changing the 18 Add 1 mL of TRIzol reagent and incubate for 2 3 minutes Scrape the plate with a sterile cell scraper and collect the slurry into a sterile RNAse free microcentrifuge tube 19 Store the cells at 8
54. tems that contain Fast thermal cycling blocks and the TaqMan hPSC Scorecard Panel 384w must be run on systems with standard thermal cycling blocks For a list of Applied Biosystems RT PCR systems compatible with TaqMan hPSC Scorecard Panels see page 6 Open the experiment template file and save a separate copy with your experimental details Run the experiment using Standard method for 384 well plates with the TaqMan Gene Expression Master Mix and Fast mode for 96 well plates with the TaqMan Fast Advanced Master Mix using the cycling parameters listed below e hw Note The experiment template files eds are available at www lifetechnologies com scorecardinstrument Refer to the appropriate instrument user guide for information on how to set up the plate document experiment or create a template from the setup file TaqMan hPSC Scorecard 384w Run mode Ramp rate Standard Step Temperature Time Cycles Hold 50 C 2 minutes Hold 95 C 10 minutes Melt 95 C 15 seconds m Anneal Extend 60 C 1 minute TaqMan hPSC Scorecard 96w FAST Run mode Ramp rate Fast Step Temperature Time Cycles Hold 50 C 20 seconds Melt 95 C 1 second m Anneal Extend 60 C 20 seconds IMPORTANT Be sure to run your qRT PCR experiment using Standard Curve method Do not use AACt comparative PCR 29 qRT PCR Using the TaqMan hPSC Scorecard Panel conti
55. ter Mixes primers specific to a pluripotency or differentiation marker or endogenous control and a TaqMan probe with a fluorescent dye label on the 5 end e g FAM or VIC dye and a minor groove binder MGB and non fluorescent quencher NFQ on the 3 end see page 37 The assays in each well are reconstituted to a 1X formulation using the compatible TaqMan master mix as described in this user guide and are designed to run under standard or Fast cycling conditions for two step RT PCR The table below lists the TaqMan hPSC Scorecard Panel and corresponding TaqMan master mix compatible with it Note that TaqMan hPSC Scorecard Kits contain the appropriate compatible TaqMan master mix which are not supplied with the individually packaged TaqMan hPSC Scorecard Panels and need to be purchased separately from Life Technologies see page 38 for ordering information TagMan hPSC Scorecard Panel Compatible TaqMan master mix TaqMan hPSC Scorecard Panel 384w TaqMan Gene Expression Master Mix for standard cycling TaqMan hPSC Scorecard Panel 96w FAST TaqMan Fast Advanced Master Mix for Fast cycling Compatible RT PCR instruments TaqMan hPSC Scorecard Panels can be used with the Applied Biosystems RT PCR systems listed below Note that TaqMan hPSC Scorecard Panel 96w FAST must be run on RT PCR systems that contain Fast thermal cycling blocks Alternately TaqMan hPSC Scorecard
56. weeks or it can be aliquoted and stored frozen at 20 C until use 1X Collagenase Type IV solution 1 mg mL for 50 mL 3 To prepare a 1 mg mL working solution of Collagenase Type IV dilute the 10X stock solution 1 10 in DMEM F 12 4 The working solution can be used for 2 weeks if properly stored at 2 8 C store in aliquots to avoid repeated warming MEF medium To prepare 100 mL of complete MEF medium aseptically mix the components listed below Complete MEF medium can be stored at 2 8 C for up to 1 week Component Volume DMEM F 12 1X with GlutaMAX I 89 mL FBS ESC Qualified 10 mL MEM Non essential Amino Acids Solution 10 mM 1mL B Mercaptoethanol 1000X 100 uL Continued on next page 31 Media and Reagents continued ESC medium MEF conditioned medium MEF CM 32 To prepare 100 mL of complete ESC medium aseptically mix the components listed below Complete ESC medium can be stored at 2 8 C for up to 1 week Component Volume DMEM F 12 1X with GlutaMAX I 79 mL KnockOut Serum Replacement KSR 20 mL MEM Non essential Amino Acids Solution 10 mM 1mL B Mercaptoethanol 1000X 100 uL Basic FGF 10 pg mL 40 pL Prepare the iPSC Medium without bFGF and then supplement with fresh bFGF to a final concentration of 4ng mL when the medium is used 1 Cover the whole surface of each new culture vessel with Attachment Factor AF solution and incubate the vessels f
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