Human Epidermal Keratinocytes User Manual
Contents
1. modified for resale or used to manufacture commercial products without prior written consent of SBI This Product should be used in accordance with the NIH guidelines developed for stem cell research SBI has pending patent applications related to the Product For information concerning licenses for commercial use contact SBI Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Limited Warranty SBI warrants that the Product meets the specifications described in this manual If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBPs liability does not extend to any damages arising from use or imprope2. 95 air humidified cell culture incubator 0 Do not disturb the culture for at least 24 hours after the culture has been initiated sac a hoe Ol D Maintaining Stock Cultures 1 Change the culture medium 24 to 36 hours after establishing the secondary culture from cryopreserved cells 2 Change the medium every other day thereafter until the culture is approximately 80 confluent Note Do not allow stock culture to exceed 80 confluent to prevent differentiation of HEKn Subculturing HEKn View the culture under the microscope to confirm that the cells are subconfluent before splitting The following protocol is designed for the subculture of one 75 cm culture flask 1 Remove all of the culture medium from the flask 2 Add 3 ml of 0 25 trypsin EDTA solution to the flask Rock the flask to ensure that the entire surfaced is covered 3 Incubate the flask at room temperature until the cells have become completely round approximately 5 10 minutes View the culture frequently under a microscope to avoid over digestion 4 Add 7mlof trypsin neutralization solution to the flask and transfer the detached cells to a sterile 15 ml conical tube 5 Centrifuge the cells at 180 g for 5 minutes 6 Remove the supernatant from the tube being careful not to dislodge the cell pellet 7 Resuspend the cells pellet in 10 ml supplemented medium Pipet the cells up and down with a 10 ml pipette to ensure a homogeneous cell Suspension 8 Determin
3. a System Biosciences Human Epidermal Keratinocytes for iPS Cell reprogramming Complete Kit Cat PC5O3hEKTN K Cells Only Cat PCSO3hHEKTN C User Manual Store kit at 80 C on receipt A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement contained in this user manual ver 121009 001 Human Epidermal Keratinocytes neonatal Cat PC503hEKTN K C Contents I Human Epidermal Keratinocytes neonatal HEKn 2 A Description ss 2 B Maintenance of HEKn __ _ __s aiwisi lt i i i_i i i i i titit s 2 C Product Information _ _ __s_ eee 4 D References ______s ss 5 E Related Products sss 5 F TechnicalSupport _ __ss eee 6 ll Licensing and Warranty Statement cc eee 7 List of Components The Human Epidermal Keratinocytes neonatal HEKn are available as either complete culture kit Cat PC503hEKTN K or cryopreserved cells Cat PC503hEKTN C in one vial Quantity Ship Ship o HEKn cells 1x10 cells vial Cryopreserved and shipped on dry ice HEKn supplemented 250 ml Store at 4 C upon receipt culture medium Trypsin neutralization 50 ml TNS Store at 4 C upon receipt solution HEKn cells 1x10 cells vial Cryopreserved and shipped on dry ice The cryopreserved HEKn cells are shipped on dry ice and shoul
4. d be immediately stored in liquid nitrogen upon receipt Properly stored cells are stable for more than 2 years from the date received 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual I Human Epidermal Keratinocytes neonatal A Description Human epidermal keratinocyte neonatal HEKn were isolated from individual neonatal foreskin Day 1 to Day 3 of age Each vial of this product contains 1X10 viable cells In our laboratory each lot of cells is performance tested by culturing the cells through multiple passages in the absence of antibiotics and antimycotics no contamination was observed during this culture period Upon thawing the cells are guaranteed to be gt 75 viable and have a potential of gt 30 population doublings when cultured according to the instructions provided in this manual B Maintenance of HEKn Cultures Culture medium We recommend serum free medium supplemented with keratinocyte growth supplement The supplemented medium included in the HEKn complete kit is complete culture medium optimized for HEKn HEKn culture medium can also be purchased from either Invitrogen Medium 154CF Cat M 154CF 500 or ATCC Cat PCS 200 030 and must be supplemented with calcium plus their respective keratincoyte Growth Supplement Invitrogen HKGS Cat S 001 5 ATCC Cat PCS 200 040 We recommend a low calcium concentration 0 05 0 07 mM to slow HEKn differentiation the calcium concen
5. e the concentration of cells in the suspension 9 Seed new culture vessels with 4 x 10 cells cm or 1 to 4 split if starting cells at a 80 confluence 10 Incubate the cultures in a 37 C 5 CO2 95 air humidified cell culture incubator Page 2 ver 1 090701 www systembio com Human Epidermal Keratinocytes neonatal Cat PC503hEKTN K C i lara HEKn Follow steps 1 6 from the Subculturing of Cells above Resuspend the cell pellet in supplemented medium Add approximately 1 ml for each T75 flask Count the number of cells and dilute the cell suspension to 2 x 10 cells ml Add an equal volume of cold 2X Freezing Media to the cell suspension Aliquot 1 ml of suspension into each cryovial 1 x 10 cells vial Place the vials in a cell freezing container and keep it at 80 C overnight Transfer the vials to a liquid nitrogen tank for long term storage NOORWD 4 C Product Information Organism Homo sapiens human Gender Male Age Neonatal Day 1 to Day 3 Morphology Cobblestone appearance Comments The serum free media is formulated to inhibit fibroblast growth and the low calcium concentration 50 70 uM slows differentiation No feeder layers or extracellular matrix proteins are required Day 2 HEKn Day 4 HEKn Day 6 HEKn 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual D References Aasen T et al 2008 Efficient and rapid generation of induced pluripotent
6. r use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2009 System Biosciences SBI Page 6 ver 1 090701 www systembio com
7. stem cells from human keratinocytes Nature Biotechnology 26 11 1276 1284 Jiang YJ et al 2009 Ceramide stimulates ABCA12 expression via peroxisom proliferator activated receptor delta in human keratinocytes J Biol Chem 284 18942 52 Carey BW et al 2009 Reprogramming of murine and human somatic cells using a single polycistronic vector Proc Natl Acad Sci 106 157 62 Takahashi K and Yamanaka S 2006 Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors Cell 126 663 676 Takahashi K et al 2007 Induction of pluripotent stem cells from adult human fibroblasts by defined factors Cell 131 861 72 Park IH et al 2008 Reprogramming of human somatic cells to pluripotency with defined factors Nature 451 141 6 E Related Products e Human Foreskin Fibroblasts neonatal as reprogramming source cell Cat PC501A HFF From mesoderm origin these neonatal HFFn are isolated from individual neonatal foreskin and therefore have a single genetic background High purity and low passage of these neonatal HFFn Day 1 to Day 3 of age provides a reliable cell source for efficient reprogramming e Human Foreskin Fibroblasts neonatal as feeder cells Cat PC502B HFF These pooled HFFs are optimized to provide a balanced nutrition to support your ES cell culture and reprogramming experiment High purity and low passage of neonatal HFFn Day 1 to Day 3 of age are characteristics of this produc
8. t Page 4 ver 1 090701 www systembio com Human Epidermal Keratinocytes neonatal Cat PC503hEKTN K C F Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 1616 North Shoreline Blvd Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual Licensing and Warranty Statement Limited Use License Use of the human epidermal keratinocytes e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions e The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use e The Product may not be resold
9. tration in the supplement medium in our kit is 0 07 mM Trypsion EDTA 0 25 trypsin EDTA Trypsin neutralization solution TNS 5 Chelexed Fetal Bovine Serum in Hanks BSS Initiating Cultures from Cryopreserved Cells To insure the highest level of viability be sure to warm medium to 37 C before using it on the cells We recommend seeding cells recovered from cryoperservation at a cell density of 4 x 10 viable cells cm or higher The procedure given below is a sample protocol for establishing cultures from the contents of one vial Prepare a bottle of supplemented keratinocyte growth medium according to the instructions supplied with that product Remove the vial from liquid nitrogen storage taking care to protect hands and eyes Dip the lower half of the vial into a 37 C water bath to thaw When the contents of the vial have thawed wipe the outside of the vial with 75 alcohol to disinfect and move the vial to a laminar flow culture hood Open the vial and pipet the cell suspension up and transfer the cells into a 15 cm conical tube with 10 ml fresh keratinocyte growth medium Pipet up and down with a 10 ml pipette to disperse the cells and centrifuge the cells at 180 g for 5 minutes Observe the cell pellet Remove the supernatant form the tube being careful not to dislodge the cell pellet Dilute the cells with fresh culture medium and seed new culture vessels with 4 x 10 cells cm Incubate the cultures in a 37 C 5 COz2
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