NeuroPrime™ Cell Kit Product Data Sheet and
Contents
1. BIOSCIENCE www essenbioscience com Neurite length measurements and IncuCyte ZOOM images obtained using NeuroLight Red infected NeuroPrime rForebrain Neurons in co culture with NeuroPrime rAstrocytes Upper left panel plots mean neurite length SD over time for all wells from a 96 well plate Images from a single well at 3 6 9 and 12 days after plating as indicated Scale Bar 200UM Safety Considerations The backbone of the Lentivirus particles in this system has been modified to improve their safety and minimize their relation to the wild type human HIV 1 virus These modifications include e The lentiviral particles are replication incompetent and only carry the non oncogenic gene of interest e A deletion in the 3 LTR AU3 resulting in self inactivation SIN of the Lentivirus after transduction and genomic integration of the target cell Yee et al 1987 Yu et al 1986 Zufferey et al 1998 This alteration renders the lentiviral genome incapable of producing package able virus following host integration e The envelope is psueudotyped with the VSV G gene from Vesicular Stomatitis Virus of the HIV 1 envelope Burns et al 1993 Emi et al 1991 Yee et al 1994 Replication defective lentiviral vectors such as the 3 generation vector provided in this product are not known to cause any diseases in humans or animals However lentivirus particles still pose some biohazardous risk because they can transduce primary h2. N ESSEN BIOSCIENCE www essenbioscience com Product Information CellPlayer NeuroPrime Cell Kit Catalog Number 4585 Contents 1x vial CellPlayer NeuroPrime rForebrain Neurons 2 x 10 cells vial 1x vial CellPlayer NeuroPrime rAstrocytes 2 x 10 cells vial 1x vial CellPlayer NeuroLight Red Lentivirus Synapsin promoter 0 45 mL vial Lot Titer Supplied by Molecular Transfer Inc Storage and Stability Cryopreserved cell vials of NeuroPrime rForebrain Neurons and NeuroPrime rAstrocytes e Transfer to liquid nitrogen immediately upon arrival e Cells will remain viable when stored in liquid nitrogen for at least 6 months from the date of receipt The NeuroLight Red Lentivirus e Transfer to 80 C freezer immediately upon receipt e Avoid repeated freeze thaw cycles e Lentivirus is stable for at least 3 months from date of receipt when stored at 80 C e Spectral Properties Ex max 588 nm Em max 633 nm Test Size Material supplied is sufficient for 1 x 96 well plate Product Description The CellPlayer NeuroPrime Cell Kit contains cryopreserved vials of forebrain neurons and astrocytes isolated from embryonic stage E18 Sprague Dawley rats as well as sufficient NeuroLight Red lentivirus to perform one 96 well experiment The lentivirus encodes a red fluorescent protein regulated by a synapsin promoter that selectively drives expression in neurons and minimizes express
3. cells mL in pre warmed NCM The cell suspension can be transferred to a sterile trough in the tissue culture hood at this point in order to facilitate pipetting of cells in the next step Using a multichannel handheld pipette dispense 100 uL of neuronal cell suspension into each well of the Poly D Lysine coated 96 well plate 15 000 neurons well CRITICAL To ensure proper mixing and uniform seeding of the neurons mix the cell suspension by gently pipetting up and down 1 2 times between seeding each row of the plate Rocking the trough is also recommended to ensure equal cell distribution Let the plate stand at room temperature in the tissue culture hood for 30 minutes and then place inside the incubator CRITICAL This step ensures the uniform distribution of cells in each well Allow cells to settle on the plate for 2 to 3 hours before proceeding Infect neurons with NeuroLight Red Lentivirus 1 2 Allow the NeuroLight Red Lentivirus to thaw on ice approximately 1 hour CRITICAL Use the equation below to calculate the volume of lentivirus required to achieve an MOI of 1 when diluted into 11 mL of NCM based on the viral titer supplied on the vial label e g for a viral titer of 5 x 10 TU mL add 0 33 mL 330 uL lentivirus to 11 mL of Neuronal culture media 11 mL Neuronal culture media viral titer 15 000 neurons per well Volume of lentivirus required mL MOI of 1 0 1 mL Based on the results of the c
4. the cell suspension at this point can result in osmotic shock and cell death Transfer the rinse media in a drop wise fashion to the 50 mL conical tube containing astrocytes while gently swirling the 50mL conical tube 9 Add 3 mL pre warmed ACM to the 50 mL conical tube in a drop wise fashion The 3mL addition should be performed slowly taking at least 1 minute 10 Centrifuge the astrocytes at 250 x g for 5 min Carefully aspirate and discard the supernatant and resuspend the cell pellet in 5 mL of ACM Using a P 1000 handheld pipet set to 800 uL triturate the cell suspension by gently aspirating and dispensing 10 15 times to ensure a single cell suspension 11 Perform a cell count e g Trypan Blue staining with hemocytometer and dilute cells to 300 000 cells mL in pre warmed ACM 12 Using a multichannel handheld pipettor plate 50 uL of astrocyte cell suspension into each well of the 96 well plate containing the cultured neurons i e 15 000 astrocytes well CRITICAL To ensure proper mixing and uniform seeding of the astrocytes mix the cell suspension by gently pipetting up and down 1 2 times between seeding each row of the plate Rocking the trough is also recommended to ensure equal cell distribution 13 Place plate into the IncuCyte ZOOM and schedule to image every 2 to 12 hours See IncuCyte ZOOM User Manual for detailed instructions on setting up an imaging schedule Day 3 Treat plate with 5 Fluoro 2 deoxyuridine and Uridin
5. alculation above dilute the appropriate amount of NeuroLight Red reagent into 11ml NCM so that an MOI of 1 is achieved when 100 uL is added to each well of the plate Mix the virus and media by pipetting up and down At 2 4 hours post plating remove plated neurons from incubator Using a multichannel pipettor add 100 uL virus solution per well at MOI 1 without mixing and return to incubator immediately CRITICAL Do not pipette up and down after adding the virus solution as this may result in damage to the plated neurons 5 8000 0280 B00 N ESSEN BIOSCIENCE www essenbioscience com Day 1 Plate astrocytes 1 16 24 hours after plating and infecting the neurons warm NCM to 37 C 2 Carefully remove 190 uL of medium per well using a multi channel pipettor and replace immediately with 140 uL of fresh pre warmed NCM Volume should now be 150 uL per well 3 Prepare 50 mL Astrocyte Culture Media 85 DMEM 15 FBS ACM by adding 7 5 mL FBS to 42 5 mL DMEM and warm to 37 C 4 Remove the vial of NeuroPrime rAstrocytes from liquid nitrogen storage and thaw in a 37 C water bath until only a tiny ice crystal remains 1 to 2 minutes 5 Wipe vial with 70 ethanol 6 In tissue culture hood use a P1000 pipettor to pre wet a tip with 1 mL ACM 7 Use the pre wetted tip to transfer the 1 mL volume of thawed astrocytes to a 50 mL conical tube 8 Rinse the cryo vial with 1 mL ACM CRITICAL Rapid addition of the media to
6. ct contains proprietary nucleic acid s coding for proprietary fluorescent protein s being including its derivatives or modifications the subject of pending patent applications and or patents owned by Evrogen JSC hereinafter Evrogen Fluorescent Proteins The purchase of Essen BioScience products incorporating these fluorescent proteins conveys to the buyer the non transferable right to use Evrogen Fluorescent Proteins only for research conducted by the buyer No rights are conveyed to modify or clone the gene encoding fluorescent protein contained in this product or to use Evrogen Fluorescent Proteins for commercial purposes The right to use Evrogen Fluorescent Proteins specifically excludes the right to validate or screen compounds for commercial purposes For information on commercial licensing contact Evrogen Licensing Department email license evrogen com For research use only Not for therapeutic or diagnostic use 9 8000 0280 B00
7. dd NeuroLight 95 media 50 media 50 media Red Lentivirus Replacement replacement replacement Plate rAstrocytes Add Uridine 5 Treatments day 6 FDUridine and beyond Begin IncuCyte scanning 2 8000 0280 B00 N ESSEN BIOSCIENCE www essenbioscience com Protocol CellPlayer NeuroPrime Cell Kit Materials required but not supplied Equipment 96 well plate flat bottom Corning Cat No 3595 or TPP Cat No 92096 Class 2 Tissue culture hood Centrifuge for pelleting cells 250 g capability inserts for 50 mL conical tubes Multichannel hand help pipettor 200 uL P 1000 handheld pipettor Cell counting system hemocytometer or automated Sterile 50 mL conical tubes for making cell dilutions Sterile troughs for preparing and containing cells and test compound dilutions Tissue culture incubator 37 C 5 CO2 humidified atmosphere IncuCyte ZOOM with CellPlayer NeuroTrack Software Module Cat No 9600 0010 Water bath set to 37 C Reagents Poly D Lysine Millipore Cat No A 003 E WEFI water Corning CellGro Mediatech Cat No 25 055 CM Neurobasal Media Life Technologies Cat No 21103049 B 27 Serum Free Supplement Life Technologies Cat No 17504044 GlutaMAX l Supplement Life Technologies Cat No 35050061 DMEM Life Technologies Cat No 11965 or 41965 Fetal Bovine Serum Sigma Aldrich Cat No F2442 or Thermo Scientific Cat No SH3007103 5 Fluoro 2 deoxyuri
8. dine Sigma Aldrich Cat No F0503 Uridine Sigma Aldrich Cat No U3003 70 ethanol w v Solutions to prepare in advance Day 1 Poly D Lysine o 100 ug mL in 12 mL of WFI quality water Day O Neuronal Culture Media for 50 mL o 48 5 mL Neurobasal Media o 0 5 mL GlutaMAx o 1mLB 27 Supplement Day 1 Neuronal Culture Media for 50 mL o 48 5 mL Neurobasal Media o 0 5 mL GlutaMAx o 1mLB 27 Supplement Astrocyte Culture Media for 50 mL o 42 5 mL DMEM O 7 5 mL FBS 3 8000 0280 B00 N ESSEN BIOSCIENCE www essenbioscience com Day 3 e 10x FdU U o Dissolve 8 mg of FdU and 28 mg of U in 100 mL Neurobasal o Aliquot and store at 20 C e 2x FdU U o Thaw 10x stock of FdU U on ice o Dilute to 2x in 12 mL of NCM in 15 mL conical tube Add 2 4 mL 10x FdU U Add 9 6 mL NCM CRITICAL Use rigorous aseptic technique at all times Only open the culture plate and medium bottles within a in a tissue culture hood Day 1 Coat 96 well plate with Poly D Lysine 1 Coat one 96 well plate with Poly D Lysine Prepare a 1x stock of Poly D Lysine final concentration 100 g mL in WFI quality water and add 100 uL to each well Replace lid and Incubate for 16 20 hours at room temperature in the tissue culture hood 2 Aspirate and discard the Poly D Lysine and rinse the plate twice with 150 uL well of WFI water If excess Poly D Lysine is not washed away it can impair neurite outgrowth 3 Leave the p
9. e CRITICAL Addition of 5 Fluoro 2 deoxyuridine and Uridine FdU U prevents proliferation of non neuronal cell types 6 8000 0280 B00 N ESSEN BIOSCIENCE www essenbioscience com 1 Remove 100 uL of media from each well using a multi channel pipette and replace with 100 uL fresh Neuronal Culture Media containing 2x concentrations of 5 Fluoro 2 deoxyuridine and uridine to a final assay concentration of 8 ug mL and 28 g mL respectively Days 6 9 12 and beyond Solutions Required 1 Neuronal Culture Media for 50 mL a 48 5 mL Neurobasal Media b 0 5 mL GlutaMAx c 1mLB 27 Supplement Feeding Cultures 1 Feed cultures with fresh NCM by performing a 50 media change To do this remove 100 uL per well and replace with 100 uL of fresh media CRITICAL Only a single FdU U treatment is required Day 3 step 1 Addition of fresh FdU U is not recommended on following days 2 Cultures can be stopped at Day 11 or continued for desired length with 50 media changes occurring every third day Example Data Use of the CellPlayer NeuroPrime Cell Kit according to the protocols described above will enable a robust and reliable assay for neurite outgrowth The figure below shows data obtained from a single 96 well plate assay over 12 days where with all wells were fed with NCM Neurite Length uM mm _ _ N o O j jo jo oa Oo 0 50 100 150 200 Time hr 7 8000 0280 B00 N ESSEN
10. ion in non neuronal cell types After six days in co culture the neurons form extensive neurite networks enabling the neurotoxic or neuroprotective effects of treatments to be assessed Dynamic changes in network length and branching are measured using IncuCyte ZOOM and the NeuroTrack software module USA EUROPE JAPAN mia Essen BioScience Inc Essen BioScience Ltd Essen BioScience K K 300 West Morgan Road BioPark Broadwater Road Cerulean Tower 15F Ann Arbor MI 48108 Welwyn Garden City Hertfordshire 26 1 Sakuragaoka cho USA AL7 3AX United Kingdom Shibuya ku Tokyo 150 8512 Japan m Phone 1 734 769 1600 44 0 1707 358688 81 3 5456 5481 essenbioscience com A ESSEN BIOSCIENCE www essenbioscience com Example Images Following the CellPlayer NeuroPrime Cell Kit protocol will ensure a neurite network length of at least 50 mm mm after ten days under control conditions using a calibrated Incucyte ZOOM instrument with optimized processing definitions 0 ym 100 0 61 x 0 53 mm 0 33 mm IncuCyte ZOOM images of the CellPlayer NeuroPrime Cell Kit after 10 days in culture Left Fluorescent image of NeuroLight Red expressing forebrain neurons in co culture with astrocytes Right Phase contrast image of neurons and astrocytes in co culture Scale bar 100um CellPlayer NeuroPrime Cell Kit Protocol Overview Day 0 Day O 4 hours Day 1 Day 3 Day 6 9 12 ef of ef of O Plate rNeurons A
11. late to dry for at least one hour with lid removed in the tissue culture hood Day 0 Thaw and plate neurons 1 Prepare the Neuronal Culture Media NCM For 50 mL of complete Neurobasal culture media add 1 mL of B 27 Serum free supplement 0 5 mL GlutaMAX I to 48 5 mL of Neurobasal media in a 50 mL conical tube 2 CRITICAL Warm NCM to 37 C prior to thawing neurons 3 Remove the vial of NeuroPrime rForebrain Neurons from liquid nitrogen storage and thaw ina 37 C water bath until only a tiny ice crystal remains 1 to 2 minutes CRITICAL Do not agitate the vial during this step 4 Wipe outside of vial with 70 ethanol 5 In tissue culture hood use a P1000 pipettor to pre wet a tip with 1 mL NCM 6 Use the pre wetted tip to transfer the 1 mL volume of thawed neuronal cells to a 50 mL conical tube 4 8000 0280 B00 10 11 12 N ESSEN BIOSCIENCE www essenbioscience com Rinse the cryo vial with 1 mL NCM and transfer the rinse media in a drop wise fashion to the 50 mL conical tube containing neurons while gently swirling the 50mL conical tube CRITICAL Rapid addition of the media at this point can result in osmotic shock and cell death The 1mL addition should take about 30 seconds In drop wise fashion add a further 2 mL pre warmed NCM to the 50 mL conical tube The 2 mL addition should take about 1 minute Perform a cell count e g Trypan Blue staining with hemocytometer and dilute neurons to 150 000
12. uarantee the absence of known and unknown infectious agents Consequently all products should always be considered potentially biohazardous and appropriate precautions should be taken Use good laboratory practice and aseptic technique at all times 8 8000 0280 B00 N ESSEN BIOSCIENCE www essenbioscience com Licenses and Warranty Essen BioScience warrants that this product performs as described on the product label and in all literature associated with the sale of said product when used in accordance with the described protocol This limited warranty is the sole warranty No other warranties exist that extend beyond this warranty either expressed or implied Essen BioScience disclaims any implied warranty of merchantability or warranty of fitness for a particular purpose Essen BioScience disclaims any responsibility for injury or damage and shall not be liable for any proximate incidental or consequential damages in connection with this product If it is proven to the satisfaction of Essen BioScience that this product fails to meet performance specifications Essen BioScience s sole obligation at the option of Essen BioScience is to replace the product or provide the purchaser with credit at or below the original purchase price This limited warranty does not extend to anyone other than the purchaser Notice of suboptimal performance must be made to Essen BioScience within 30 days of receipt of the product This Essen BioScience produ
13. uman cells and can integrate into the host cell genome thus posing some risk of insertional mutagenesis For this reason we highly recommend that you treat lentiviral stocks as Biosafety Level 2 BSL 2 BL 2 organisms and strictly follow all published BL 2 guidelines with proper waste decontamination For more information about the BL 2 guidelines and Lentivirus handling we recommend referring to local documentation based on geography The Essen BioScience 3 generation HIV based lentivirus meet BL 2 requirements based on the criteria in the document Biosafety in Microbiological and Biomedical Laboratories 5 Edition published by the Centers for Disease Control CDC This document may be downloaded at http www cdc gov biosafety publications bmbI5 index htm Institutional Guidelines Safety requirements for use and handling of lentiviruses may vary at individual institutions We recommend consulting your institution s health and safety guidelines and or officers prior to implementing the use of these reagents in your experiments A detailed discussion of lentiviral vectors is provided in Pauwels K et al 2009 State of the art lentiviral vectors for research use Risk assessment and biosafety recommendations Curr Gene Ther 9 459 474 Biohazard Note The NeuroPrime rForebrain Neurons and rAstrocytes contain cells of rodent origin Although the cells test negative for mycoplasma bacteria and fungi no test procedure can g
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