GIBCO Human NSCs - Thermo Fisher Scientific
Contents
1. Additional The products listed in this section may be used with GIBCO Products hNSCs For more information refer to our website www invitrogen com or contact Technical Support see page 25 Item Quantity Cat no StemPro NSC SFM contains KnockOut DMEM F 12 FGF Basic Recombinant Human EGF Recombinant Human and 1 kit A1050901 StemPro Neural Supplement StemPro Neural Supplement 10 mL A10508 01 GlutaMAX I Supplement 100 mL 35050 061 KnockOut DMEM F 12 500 mL 12660 012 FGF Basic Recombinant Human bFGF 10 pg PHG0024 EGF Recombinant Human 10 ug PHG0314 Fetal Bovine Serum FBS ES Cell Qualified 500 mL 16141 079 BSA 10 Stock Solution 25 mL P2489 Dulbecco s Phosphate Buffered Saline D PBS containing no 500 mL 14190 144 calcium magnesium or phenol red Dulbecco s Phosphate Buffered Saline D PBS containing 500 mL 14040 133 calcium and magnesium but no phenol red Dulbecco s Modified Eagle Medium D MEM 1X liquid 1000 mL 11995 040 high glucose CELLStart Defined Humanized Substrate for Cell Culture 2mL A1014201 Geltrex Reduced Growth Factor Basement Membrane 5 mL 12760 021 Matrix Fibronectin Human Plasma 5 mg 33016 015 Laminin Natural Mouse Img 23017 015 StemPro Accutase Cell Dissociation Reagent 100 mL A11105 01 TrypLE Express 1X liquid without phenol red 100 mL 12604 013 Neurobasal Medium 1X liquid 500 mL 21103 049 B 27 S2. hNSCs to differentiate into Differentiation neurons oligodendrocytes and astrocytes by exposing the Protocol cells to specific factors For more information on how to enrich GIBCO hNSCs toward neurons oligodendrocytes and astrocytes upon selection on the appropriate differentiation medium contact Technical Support page 25 17 Characterizing the Phenotype of GIBCO Human NSCs Introduction This section provides information on phenotypic marker expression of GIBCO hNSCs in their undifferentiated state and after their differentiation into neurons oligodendrocytes and astrocytes Phenotypic The following table lists the primary antibodies used for Markers classifying undifferentiated GIBCO hNSCs neurons oligodendrocytes and astrocytes See page 24 for ordering information Antigen Dilution Antibody ratio type Undifferentiated GIBCO Nestin Abcam Cat no 1 1 000 Rabbit IgG hNSCs Ab5968 SOX2 R amp D Systems 1 200 Mouse IgG Cat no MAB2018 Neurons MAP2 1 200 IgG kappa Dcx 1 400 Rabbit IgG Oligodendrocytes GalC Millipore Cat no 1 200 Mouse IgG MAB342 A2B5 1 100 Mouse IgM Astrocytes CD44 1 50 Mouse IgG GFAP 1 200 Rabbit IgG See Figures 1 and 2 on pages 2 3 for examples of fluorescent Note images showing phenotypic marker expression of GIBCO 18 hNSCs in their undifferentiated state and after their differentiation into neurons oligodendrocytes and astr
3. Stem Cells H9 Derived and StemPro Neural Supplement are shipped on dry ice FGF Basic Recombinant Human and EGF Recombinant Human are shipped on gel ice KnockOut DMEM F 12 is shipped at room temperature Kit components and storage conditions for N7800 100 and N7800 200 are listed in the table below N7800 100 Amount Storage GIBCO Human Neural Stem Cells H9 Derived 1mL Liquid gt 1 x 10 cells mL in freezing medium nitrogen N7800 200 Amount Storage GIBCO Human Neural Stem Cells H9 Derived 1mL Liquid gt 1 x 106 cells mL in freezing medium nitrogen KnockOut DMEM F 12 500 mL 2 to 8 C in the dark StemPro Neural Supplement 10 mL 5 to 20 C in the dark FGF Basic Recombinant Human 10 ug 2 to 8 C EGF Recombinant Human 10 pg 2 to 8 C Composition of the freezing medium is proprietary Intended Use Handle cells as potentially biohazardous material under at least Biosafety Level 1 BL 1 containment This product contains Dimethyl Sulfoxide DMSO a hazardous material Review the Safety Data Sheet SDS before handling Safety Data Sheets SDSs are available on our website at www invitrogen com sds GIBCO Human Neural Stem Cells H9 Derived are for research use only They are not intended for any animal or human therapeutic or diagnostic use GIBCO Human Neural Stem Cells H9 Derived Neural Stem Cells GIBCO Human Neural Stem Cells H9 Derived Character
4. Zhao C Deng W and Gage F H 2008 Mechanisms and functional implications of adult neurogenesis Cell 132 645 660 2009 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 28 invitrogen Corporate Headquarters 5791 Van Allen Carlsb T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
5. bmbl5 bmbl5toc htm Guidelines for Follow the general guidelines below to grow and maintain Culturing GIBCO hNSCs GIBCO e All solutions and equipment that come in contact with Human NSCs the cells must be sterile Always use proper aseptic technique and work in a laminar flow hood e Before starting experiments make sure that the cells have been established at least 1 passage e For consistent results in your differentiation studies and other experiments we recommend using cells below passage 3 P3 If you expand GIBCO hNSCs beyond P3 we recommend that you perform another round of characterization prior to further experiments e When thawing or subculturing cells transfer cells into pre warmed medium Thaw hNSCs rapidly but resuspend them slowly to avoid osmotic shock e For general maintenance of GIBCO hNSCs in adherent culture the cells should be approximately 90 confluent prior to subculturing Passage the cells at a seeding density of 50 000 cells cm Note Passaging hNSCs at a lower density decreases their proliferation efficiency e You may culture GIBCO hNSCs on tissue culture vessels coated with CELLStart Geltrex fibronectin or a double coating of poly L ornithine and laminin Note The attachment strength of GIBCO hNSCs is greatest for CELLStart followed by fibronectin and is weakest for poly L ornithine e Standard physical conditions for GIBCO hNSCs grown in StemPro NSC
6. neurons oligodendrocytes and astrocytes e Stain positive for the neural stem cell type specific markers nestin and SOX2 and the proliferation marker Ki67 gt 80 e Stain lt 5 for embryonic stem cell specific marker Oct4 e Exhibit a doubling time of 40 50 hours e Retain their proliferation and differentiation potential for at least 3 passages after thawing Continued on next page Phenotype Marker Expression of GIBCO Human NSCs H9 Derived Undifferenti The presence of basic fibroblast growth factor bFGF in ated GIBCO complete StemPro NSC SEM allows the maintenance of Human NSCs GIBCO hNSCs in their undifferentiated state The images below show the phenotype marker expression of undifferentiated human NSCs after three rounds of passaging P3 in StemPro NSC SFM Figure 1 Fluorescence image 20X of GIBCO hNSCs at P3 that have been cultured in StemPro NSC SEM and stained for the NSC phenotype markers nestin green and the proliferation marker Ki67 red Cell nuclei were counterstained with DAPI blue Approximately 90 of the cells stain positive for the undifferentiated NSC marker nestin and the proliferation marker Ki67 Lack of Oct4 staining indicates that there are no remnant hESCs in the culture data not shown Continued on next page Phenotype Marker Expression of GIBCO Human NSCs continued Differentiation GIBCO hNSCs spontaneously differentiate into neurons Potential
7. of oligodendrocytes or astrocytes upon withdrawal of bFGF and GIBCO EGF from culture media Alternatively they can be enriched Human NSCs toward a specific lineage upon selection on differentiation medium see Figure 2 below Figure 2 Fluorescence images 20X of GIBCO hNSCs that have been cultured in StemPro NSC SEM for three passages and then allowed to differentiate into neurons oligodendrocytes or astrocytes Upon directed differentiation cells start to lose the undifferentiated NSC marker nestin but stain positive for the differentiated cell type markers Dex GalC and GFAP Cells were stained for the undifferentiated NSC markers nestin red and SOX2 green prior to directed differentiation panel A Cell were then differentiated into neurons and glial cells and respectively stained for the neuronal marker Dex green panel B for the oligodendrocyte marker GalC red panel C or for the astrocyte marker GFAP green panel D The nuclei were counterstained with DAPI blue in panels B D Methods Handling GIBCO Human NSCs As with other mammalian cell lines handle GIBCO Human Neural Stem Cells as potentially biohazardous material under at least Biosafety Level 1 BL 1 containment For more information on BL 1 guidelines refer to Biosafety in Microbiological and Biomedical Laboratories 5 ed published by the Centers for Disease Control or see the following website www cdc gov od ohs biosfty
8. solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Life Technologies Corporation 5
9. solution 500X Aliquot the solution and store it at 20 C until use Thaw the laminin slowly at 2 8 C and prepare 10 ug mL working solution in cell culture grade distilled water Aliquot the working solution into polypropylene tubes and store the tubes at 20 C until use Avoid repeated freeze thaw cycles Note Laminin may form a gel if thawed too rapidly Dilute the poly L ornithine stock solution 1 500 in cell culture grade distilled water to make 20 ug mL working solution Coat the surface of the culture vessel with or without cover slips with the poly L ornithine working solution 14 mL for T75 7 mL for T25 3 5 mL for 60 mm dish 2 mL for 35 mm dish Incubate the culture vessel overnight at 4 C or for 1 hour at 37 C Rinse the culture vessel twice with sterile water Coat the surface of the culture vessel with or without cover slips with the laminin working solution 14 mL for T75 7 mL for T25 3 5 mL for 60 mm dish 2 mL for 35 mm dish Incubate the culture vessel overnight at 4 C or for 2 hours at 37 C Rinse the culture vessel with D PBS without calcium or magnesium see page 23 and store the vessel covered with D PBS until use Immediately before use remove all D PBS and replace it with complete StemPro NSC SFM Note You may coat the plates in advance and store them at room temperature wrapped tightly with Parafilm for up to 1 week Do not remove D PBS until just prior to use Make sure th
10. with Parafilm for up to 2 weeks Do not remove CELLStart solution until just prior to use Make sure the plates do not dry out Thaw the Geltrex bottle at 4 C overnight to prevent polymerization The next day dilute Geltrex 1 2 with D MEM F 12 at 4 C to make 100X stock solution using an ice bucket to keep the bottles cold Quickly prepare 0 5 mL aliquots in 50 mL conical tubes pre chilled on ice and store the tubes at 20 C Thaw 1 tube of Geltrex 0 5 mL aliquoted as above slowly at 4 C and add 49 5 mL of cold D MEM F 12 1 100 dilution Mix gently Cover the whole surface of each culture plate with the Geltrex solution 1 5 mL for a 35 mm dish 3 mL for 60 mm dish 5 mL for a T25 culture flask Seal each dish with parafilm to prevent drying and incubate 1 hour at room temperature in a laminar flow hood Remove the vessel from the incubator and store it until use Immediately before use remove all Geltrex solution wash once with D PBS with calcium and magnesium and replace pre warmed complete medium Note You may store the Geltrex treated dish at 4 C for up to 1 month Do not remove Geltrex solution until just prior to use Continued on next page Preparing Matrix for Adherent Cell Culture continued Coating Culture 1 Vessels with Poly L Ornithine and Laminin Dissolve poly L ornithine Sigma Cat no P3655 in cell culture grade distilled water to make 10 mg mL stock
11. 791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com 27 References Bjorklund A and Lindvall O 2000 Cell replacement therapies for central nervous system disorders Nat Neurosci 3 537 544 Gage F H 2000 Mammalian neural stem cells Science 287 1433 1438 Shin S and Vemuri M 2010 Culture and Differentiation of Human Neural Stem Cells In Protocols for Neural Cell Culture Springer Protocols Handbooks 4 edn Doering Laurie C ed Humana Press pp 51 74 Shin S Sun Y Liu Y Khaner H Svant S Cai J Xu Q X Davidson B P Stice S L Smith A K Goldman S A Reubinoff B E Zhan M Rao M S Chesnut J D 2007 Whole genome analysis of human neural stem cells derived from embryonic stem cells and stem and progenitor cells isolated from fetal tissue Stem Cells 25 1298 1306 Shin S Mitalipova M Noggle S Tibbitts D Venable A Rao R Stice S L 2006 Long term proliferation of human embryonic stem cell derived neuroepithelial cells using defined adherent culture conditions Stem Cells 24 125 138 Shin S Rao M S 2006 Large scale analysis of neural stem cells and progenitor cells Neurodegener Dis 3 106 111 Temple S 2001 The development of neural stem cells Nature 414 112 117 Wu Y Y Mujtaba T and Rao M S 2002 Isolation of stem and precursor cells from fetal tissue Methods Mol Biol 198 29 40
12. CELLStart up to 1 50 for better adhesion Incubation for poly L ornithine too short Make sure you incubate your culture vessel overnight at room temperature after coating it with poly L ornithine Differen The table below lists some potential problems and solutions that tiating Cells help you troubleshoot your differentiation problems Problem Cause Solution Cells fail to Culture medium Remove bFGF from culture medium differentiate contains bFGF Cell density too Reduce cell density high and endogenous bFGF is preventing differentiation Cells have been passaged too many times Obtain new GIBCO hNSCs 21 Appendix Recipes Para To prepare 20 paraformaldehyde PFA stock solution formaldehyde 1 Add PBS to 20 g of EM grade paraformaldehyde Solution Electron Microscopy Services Cat no 19208 and bring the volume up to 100 mL 2 Add 0 25 mL of 10 N NaOH and heat the solution at 60 C using a magnetic stirrer until the solution is completely dissolved 3 Filter the solution through a 0 22 micron filter and cool on ice Make sure the pH is 7 5 8 0 4 Aliquot 2 mL in 15 mL tubes freeze the tubes on dry ice and store them at 20 C To prepare 4 PFA for fixing 1 Add 8 mL PBS into each 15 mL tube containing 2 mL of 20 PFA and thaw each tube in a 37 C water bath 2 Once the solution has dissolved the tubes cool on ice 22 Additional Products
13. Detach the cells following steps 4 6 of the subculture procedure pages 13 14 Transfer the cells into a 15 mL or 50 mL sterile conical tube Centrifuge the cells at 200 x g for 4 minutes at room temperature Aspirate the medium and discard Resuspend the cell pellet in a minimal volume of pre warmed complete StemPro NSC SFM and remove a sample for counting Determine the viable cell density and calculate the required volume of freezing medium to give the cells the desired final freezing cell density i e 1 0 x 10 cells mL 1 mL vial Centrifuge the cells at 200 x g for 4 minutes at room temperature Gently aspirate the medium and discard Resuspend the pellet using complete StemPro NSC SFM to half the final freezing volume Note The cell concentration is 2 x 10 cells mL at this stage of the freezing procedure Add the same amount of 2X freezing medium to the resuspended cells in a drop wise manner Note The final concentration of DMSO in 1X freezing medium is 10 and the final cell concentration is 1 x 10 cells mL Transfer 1 mL 1 x 106 cells aliquots of the cell suspension into cryovials Achieve cryopreservation overnight in a controlled rate freezing apparatus following standard procedures 1 C decrease per minute The next day transfer the frozen vials to a liquid nitrogen tank vapor phase for long term storage Note You may check the viability and recovery of frozen cells 24 hours after storing c
14. I 4 6 diamidino 2 phenylindole dihydrochloride 10 mg D1306 ProLong Gold Antifade Reagent 10 mL P36930 ProLong Gold Antifade Reagent with DAPI 10 mL P36931 24 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for Technical resources including manuals vector maps and sequences application notes SDSs FAQs formulations citations handbooks etc e Complete Technical Support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan Minato ku 3 Fountain Drive Tel 1 760 603 7200 Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail E mail E mail tech_support invitrogen com jpinfoGinvitrogen com eurotech invitrogen com Safety Data Sheets SDSs Certificate of Analysis Safety Data Sheets SDSs are available on our website at www invitrogen com sds The Certificate of Analysis provides detailed quality control information for each p
15. SC SFM Preparing StemPro NSC SFM complete medium consists of KnockOut Complete D MEM F 12 with StemPro Neural Supplement EGF bFGF StemPro NSC and GlutaMAX I Complete medium is stable for up to 4 SFM weeks when stored in the dark at 4 C To make 100 mL of complete StemPro NSC SFM aseptically mix the following components Component Concentration Amount KnockOut D MEM F 12 1X 97 mL GlutaMAX I Supplement 2 mM 1mL bFGF 20 ng mL 2 ug EGF 20 ng mL 2 ug StemPro Neural Supplement 2 2mL Note You may observe a white precipitate when thawing StemPro Neural Supplement This precipitate will disappear when the supplement is completely thawed or dissolved Preparing Matrix for Adherent Cell Culture Coating Culture 1 Vessels with CELLStart Coating Culture Vessels with Geltrex Dilute CELLStart 1 100 in D PBS with calcium and magnesium i e 50 uL of CELLStart into 5 mL of D PBS see page 23 Coat the surface of the culture vessel with the working solution of CELLStart 14 mL for T75 7 mL for T25 3 5 mL for 60 mm dish 2 mL for 35 mm dish Incubate the culture vessel at 37 C in a humidified atmosphere of 5 CO in air for 1 hour Remove the vessel from the incubator and store it until use Immediately before use remove all CELLStart solution and replace it with complete StemPro NSC SFM Note You may coat the plates in advance and store them at 4 C wrapped tightly
16. SFM are 36 to 38 C in a humidified atmosphere of 4 to 6 CO in air Media Requirements Important Media Requirements It is very important to strictly follow the guidelines for culturing GIBCO hNSCs in this manual to keep them undifferentiated We recommend using complete StemPro NSC SEM for optimal growth and expansion of GIBCO hNSCs and to keep the NSCs undifferentiated StemPro NSC SEM is designed to support growth of neural stem cells derived from embryonic stem cells or isolated from fetal tissue as adherent culture on CELLStart Geltrex fibronectin or poly L ornithine laminin coated tissue culture treated vessels see page 23 for ordering information e Prepare your growth medium prior to use e To maintain undifferentiated NSCs supplement the medium every day with bFGF to 10 ng mL Note If you are using complete StemPro NSC SFM to culture your cells you do not need to supplement the medium with bFGF e When thawing or subculturing NSCs transfer them into pre warmed medium at 37 C e We recommend that you aliquot complete growth medium into required working amounts to avoid exposing the medium to 37 C multiple times e You may store the complete StemPro NSC SFM in the dark at 4 C for up to four weeks Do not freeze complete StemPro NSC SFM e You may refreeze unused StemPro Neural Supplement however avoid repeated freeze thaw cycles Preparing Complete StemPro N
17. and 90 humidity For optimal performance and cell growth re feed the culture with fresh medium every two days Freezing GIBCO Human NSCs Materials Needed Guidelines Culture vessels containing GIBCO Human NSCs Complete StemPro NSC SFM DMSO use a bottle set aside for cell culture open only in a laminar flow hood Disposable sterile 15 mL or 50 mL conical tubes D PBS containing no calcium magnesium or phenol red Dissociation reagent TrypLE or StemPro Accutase pre warmed to 37 C Hemocytometer cell counter and Trypan Blue or the TM Countess Automated Cell Counter Sterile freezing vials Controlled rate freezing apparatus e g Mr Frosty Liquid nitrogen storage container When freezing GIBCO Human NSCs we recommend the following Freeze cells at a density of 1 x 10 viable cells mL and a volume of 1 mL vial Use a 2X freezing medium composed of 80 complete StemPro NSC SFM and 20 DMSO Bring the cells into the freezing medium in two steps as described in this section Continued on next page 15 Freezing GIBCO Human NSCs continued Freezing Procedure 16 10 11 12 Prepare 2X freezing medium of 80 complete StemPro NSC SFM and 20 DMSO Keep the freezing medium on ice until use Aspirate the spent complete StemPro NSC SFM from the culture vessel Wash the cells with D PBS without Ca and Mg Aspirate and discard the D PBS
18. e and evenly distribute it over the attached cell layer Incubate the cells for 2 to 5 minutes at room temperature TM Once you observe cell detachment detached cells will move with tilting of the flask gently pipet the cells up and down to break the larger clumps into a single cell suspension Protocol continued on next page Continued on next page 13 Subculturing GIBCO Human NSCs continued Passaging GIBCO Human NSCs continued 14 Protocol continued from previous page 6 10 Th 12 Stop the cell dissociation reaction by adding 9 mL of complete StemPro NSC SFM Disperse the medium by pipetting it over the cell layer surface several times Transfer the cells to a 15 mL or a 50 mL conical tube and centrifuge the tube at 200 x g for 4 minutes at room temperature Aspirate and discard the medium Resuspend the cell pellet in a minimal volume of pre warmed complete StemPro NSC SEM and remove a sample for counting Determine the total number of cells and percent viability using your method of choice Following the coating procedure pages 7 9 remove the coating solution from each coated culture vessel and replace the solution with 5 mL of complete StemPro NSC SFM Add enough cell suspension to each coated culture vessel to provide 5 x 104 cells per cm e g 1 25 x 106 cells perT25 flask Ensure an even distribution of the cell suspension Incubate the cells at 37 C 5 CO
19. e approximately 1 drop per second while swirling the tube 4 Addan additional 5 mL of pre warmed complete StemPro NSC SFM to the same tube 5 Toremove the cryoprotectant centrifuge the cells for 4 minutes at 200 x g and aspirate the supernatant 6 Resuspend the cells in 2 mL of complete StemPro NSC SFM 7 Determine the viable cell count using your method of choice The total number of viable cells should be gt 1 x 10 8 Plate the resuspended cells at a seeding density of 1 0 x 105 cells per cm on a CELLStart Geltrex fibronectin or poly L ornithine laminin coated tissue culture treated culture plate 9 Incubate the plate at 37 C 5 CO and 90 humidity and allow cells to adhere for at least 24 hours 10 The next day replace the medium with an equal volume of fresh pre warmed complete StemPro NSC SFM 11 In 4 7 days when the culture is 90 confluent you may proceed to passage your GIBCO hNSCs If you are culturing GIBCO hNSCs in growth medium other than complete StemPro NSC SFM make sure to supplement the medium every day with bFGF to 20 ng mL to maintain your cells undifferentiated Important Continued on next page 11 Thawing and Establishing GIBCO Human NSCs continued Expected The total number of viable GIBCO hNSCs should be Results gt 1x 10 after thawing Figure 3 Phase contrast images 10X of GIBCO hNSCs cultured in StemPro NSC SFM at day 1
20. e plates do not dry out Continued on next page Preparing Matrix for Adherent Cell Culture continued Coating Culture Using fibronectin as a matrix Vessels with 1 Fibronectin Dilute fibronectin see page 23 in distilled water to make 1 mg mL stock solution Store the solution at 20 C Dilute fibronectin stock solution 1 50 in PBS see page 23 to make 20 pg mL working solution Store the solution at 20 C until use Coat the surface of the culture vessel with the working solution of fibronectin 14 mL for 175 7 mL for T25 3 5 mL for 60 mm dish 2 mL for 35 mm dish Incubate the culture vessel at 37 C in a humidified atmosphere of 5 CO in air for 1 hour Remove the vessel from the incubator and store it until use Immediately before use remove all fibronectin solution and replace it with complete StemPro NSC SFM Note You may coat the plates in advance and store them at 4 C wrapped tightly with Parafilm for up to 2 weeks Do not remove the fibronectin solution until just prior to use Make sure the plates do not dry out Thawing and Establishing GIBCO Human NSCs Materials Needed Note 10 e GIBCO hNSCs stored in liquid nitrogen e Ethanol or 70 isopropanol e Complete StemPro NSC SEM see page 6 pre warmed to 37 C e Disposable sterile 50 mL tubes e 37 C water bath e 37 C incubator with a humidified atmosphere of 5 CO e Microcentrifuge e 35 mm tissue culture
21. erum Free Supplement 50X liquid 10 mL 17504 044 N 2 Supplement 100X liquid 5mL 17502 048 Antibiotic Antimycotic 100X liquid 100 mL 15240 062 Continued on next page 23 Additional Products continued Additional The products listed in this section may be used with GIBCO Products hNSCs For more information refer to our website continued www invitrogen com or contact Technical Support see page 25 Item Quantity Cat no Trypan Blue Stain 100 mL 15250 061 LIVE DEAD Cell Vitality Assay Kit 1000 assays L34951 Countess Automated Cell Counter includes 50 Countess 1 unit C10227 cell counting chamber slides and 2 mL of Trypan Blue Stain Water distilled 20 x 100 mL 15230 196 Products for The products listed below may be used for analyzing the Marker phenotype of undifferentiated GIBCO hNSCs neurons Analysis oligodendrocytes and astrocytes In addition to the primary antibodies listed below Invitrogen offers a variety of isotype specific secondary antibodies conjugated with enzymatic and fluorescent indicators and antibody sera and diluents For more information refer to www invitrogen com or contact Technical Support see page 25 Item Quantity Cat no Mouse anti MAP2 100 ug 13 1500 Mouse anti human CD44 0 5 mL MCHD4400 Rabbit anti Doublecortin Dcx 100 ug 48 1200 Mouse anti A2B5 105 100 ug 433110 ewen LAR Glial Fibrillary Acid Protein 1mL 18 0063 DAP
22. invitrogen GIBCO Human Neural Stem Cells H9 hESC Derived Catalog nos N7800 100 N7800 200 Rev date 7 December 2009 Manual part no A11592 MAN0001758 Table of Contents Contents and Storage entente tee nte tete te entree ea rvek ERA iv GIBCO Human Neural Stem Cells H9 Derived sss 1 Phenotype Marker Expression of GIBCO Human NSCs H9 Derived 2 L2 c M 4 Handling GIBCO Human NSCs Media Requirements usen peer pi Preparing Complete StemPro NSC SFM seen 6 Preparing Matrix for Adherent Cell Culture 7 Thawing and Establishing GIBCO Human NSCS sss 10 Subculturing GIBCO Human NSCS sse 13 Freezing GIBCO Human NSC8 sneskred 15 Differentiating GIBCO Human NSCs sssssseseeeeeeeeneee 17 Characterizing the Phenotype of GIBCO Human NSCS sss 18 Troubleshooting eee ttes ite e rer leiten 20 Appendix kerika ae tees eet A deke rn de nae euge ur 22 REGI DOS a erste eee ce p esu edu ees D MES 22 Additional Products ether i t ei res 23 Technical S ppOtt 5i uere aee RHODE FANE 25 Purchaser Notification eee eee 26 REPETEN COS 5 c seh eis enit eiecti unie hs ci ME N LE 28 Contents and Storage Kit Configurations Shipping Kit Contents and Storage Catalog no N7800 100 includes cells only Catalog no N7800 200 includes cells plus media GIBCO Human Neural
23. istics of GIBCO Human Neural Stem Cells H9 Derived Neural stem cells NSCs are self renewing multipotent stem cells of nervous system which can differentiate into neurons oliogdendrocytes and astrocytes Multipotent NSCs can be isolated from the fetal or adult central nervous system or derived from embryonic stem cells NSCs are an invaluable resource not only for neuroscience and stem cell studies such as regulation of neurogenesis neurotransmitter and receptor functions and stem cell differentiation but also for tissue engineering cell and genetic therapy and transplantation experiments to treat neurodegenerative diseases and neurological disorders GIBCO Human Neural Stem Cells H9 Derived are derived from the NIH approved H9 WA09 human embryonic stem cells hESCs Each vial of GIBCO hNSCs contains gt 1 x 10 viable cells that can be propagated as an adherent culture in complete StemPro NSC SFM see page 6 for composition GIBCO Human NSCs hNSCs retain their normal female human karyotype and their potential to differentiate into neurons and glial cells after multiple passages GIBCO hNSCs remove the complicated isolation or derivation process and allow the researchers who do not have access to human embryonic stem cells or human tissue the use of the hNSCs Derived from the NIH approved H9 WA09 human embryonic stem cells e Retain their capacity for self renewal e Can differentiate into
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25. ocytes Continued on next page Characterizing the Phenotype of GIBCO Human NSCs continued Immunocyto Fixing Cells chemistry 1 Remove culture medium and gently rinse the cells once with D PBS without dislodging the cells 2 Fix the cells with 4 fresh Paraformaldehyde Fixing Solution PFA see Appendix page 22 for recipe at room temperature for 15 minutes 3 Rinse3X with D PBS containing Ca and Mg Check for the presence of cells after fixing Proceed to staining described below You may store slides for up to 3 4 weeks in D PBS at 4 C before staining Do not allow slides to dry Staining Cells 1 Incubate cells for 30 60 minutes in blocking buffer 5 serum of the secondary antibody host species 1 BSA 0 1 Triton X in D PBS with Ca and Mg Note If you are using a surface antigen such as GalC omit Triton X from the blocking buffer 2 Remove the blocking buffer and incubate the cells overnight at 4 C with primary antibody diluted in 5 serum Ensure that the cell surfaces are covered uniformly with the antibody solution 3 Wash the cells 3X for 5 minutes with D PBS containing Ca and Mg if using a slide use a staining dish with a magnetic stirrer 4 Incubate the cells with fluorescence labeled secondary antibody 5 serum in D PBS with Ca and Mg in the dark at 37 C for 30 45 minutes 5 Wash the cells 3X with D PBS containing Ca and Mg and in the last wash c
26. ounter stain the cells with DAPI solution 3 ng mL for 5 minutes and rinse with D PBS 6 If desired mount using 3 drops of ProLong Gold antifade reagent per slide and seal with the cover slip see page 24 for ordering information You may store the slides in the dark at 4 C 19 Troubleshooting Culturing Cells Problem The table below lists some potential problems and solutions that help you troubleshoot your cell culture problems Cause Solution No viable cells after thawing stock Stock not stored correctly Order new stock and store in liquid nitrogen Keep in liquid nitrogen until thawing Home made stock not viable Freeze cells at a density of 1 x 10 viable cells mL Use low passage cells to make your own stocks Follow procedures in Thawing and Establishing GIBCO Human NSCs page 11 and Freezing GIBCO Human NSCs page 16 exactly Slow freezing and fast thawing is the key Add Freezing Medium in drop wise manner slowly At time of thawing thaw quickly and do not expose vial to the air but quickly change from nitrogen tank to 37 C water bath Obtain new GIBCO Human NSCs Thawing medium not correct Use pre warmed complete StemPro NSC SFM prepared as described on page 6 Cells too diluted Generally we recommend a high density culture of 1 x 10 cells per cm at the time of recovery Cell not handled gently GIBCO Human NSCs are f
27. panel A and at day 3 panel B after thawing 12 Subculturing GIBCO Human NSCs Materials Needed Passaging GIBCO Human NSCs Culture vessels containing GIBCO hNSCs 90 confluent CELLStart Geltrex fibronectin or poly L ornithine and laminin coated tissue culture treated flasks plates or dishes see pages 7 9 Complete StemPro NSC SFM pre warmed to 37 C see page 6 Disposable sterile 15 mL or 50 mL conical tubes 37 C incubator with humidified atmosphere of 5 CO Dulbecco s Phosphate Buffered Saline D PBS containing no calcium magnesium or phenol red Dissociation reagent TrypLE or StemPro Accutase pre warmed to 37 C Hemocytometer cell counter and Trypan Blue or the TM Countess Automated Cell Counter Observe the GIBCO hNSC culture under the microscope to confirm that the cells are 90 confluent and ready to be passaged Pre warm the cell dissociation reagent TrypLE or StemPro Accutase and the complete StemPro NSC SFM to 37 C before use 1 2 Aspirate the spent complete medium from the cells Rinse the surface of the cell layer with D PBS without Ca and Mg approximately 2 mL D PBS per 10 cm culture surface area by adding the D PBS to the side of the vessel opposite the attached cell layer and rocking back and forth several times Aspirate and discard the D PBS To detach the cells add 1 mL of pre warmed TrypLE or StemPro Accutas
28. ragile treat your cells gently do not vortex bang the flasks to dislodge the cells or centrifuge the cells at high speeds Do not expose neurons to air Poly L ornithine incompletely removed from culture vessel Poly L ornithine is toxic to cells Completely remove poly L ornithine from the culture vessel by washing the vessel twice with PBS without Ca and Mg Cells grow slowly Growth medium not correct Use pre warmed complete StemPro NSC SFM Cells passaged gt 3 times Use healthy NSCs under passage 4 i e 3 passages after thawing do not overgrow 20 Continued on next page Troubleshooting continued Culturing Cells continued Problem Cause Solution Cells Culture Thaw and culture fresh vial of new GIBCO differentiated conditions not hNSCs Follow thawing instructions page 11 correct and subculture procedures pages 13 14 exactly Do not omit bFGF from the medium Cell seeding Cells passaged too sparsely or cells allowed to density at the time get too confluent can cause differentiation of plating is too Seed cells at a density of 0 5 x 10 cells cm low or too high for adherent cultures Cells not Used D PBS Be sure to prepare CELLStart coated culture adherent after without Ca and vessels using D PBS containing Ca and Mg initial thaw Mg for see page 23 for ordering information CELLStart CELLStart too You may increase the concentration of dilute
29. roduct Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box 25 Purchaser Notification Limited Warranty 26 Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the co
30. ryovials in liquid nitrogen by following the procedure outlined in Thawing and Establishing GIBCO Human NSCs page 10 Differentiating GIBCO Human NSCs Introduction One critical hallmark of NSCs is their ability to differentiate into neurons and glial cells Traditional and modern bioassays are used to demonstrate the multipotency of GIBCO hNSCs to differentiate along these lineages This section provides guidelines for spontaneously differentiating GIBCO hNSCs Materials In addition to materials for passaging GIBCO hNSCs see Needed page 13 the following materials are required e StemPro NSC SFM without the growth factors i e without bFGF and EGF e CELLStart fibronectin or poly L ornithine laminin coated tissue culture treated plate see pages 7 9 Spontaneous To spontaneously differentiate GIBCO hNSCs into neurons Differentiation oligodendrocytes and astrocytes Protocol 1 Plate GIBCO hNSCs on a CELLStart fibronectin or poly L ornithine coated tissue culture treated plate at 2 5 x 104 cells cm following the protocol for passaging GIBCO hNSCs see pages 13 14 2 After 2 days change medium to StemPro NSC SFM without the growth factors i e withdraw growth factors from cell culture and replace the medium with fresh medium every 2 to 3 days Do not expose cells to air at any time after they have Important differentiated into neurons Directed You may also induce GIBCO
31. treated plate coated with CELLStart Geltrex fibronectin or poly L ornithine and laminin see pages 7 9 Note We recommend thawing GIBCO hNSCs onto 35 mm plates to maximize the efficiency of recovery e Hemocytometer cell counter and Trypan Blue or the TM Countess Automated Cell Counter TM The Countess Automated Cell Counter is a benchtop instrument designed to measure cell count and viability live dead and total cells accurately and precisely in less than a minute per sample using the standard Trypan Blue technique see page 24 for ordering information Using the same amount of sample that you currently use with the hemocytometer the Countess Automated Cell Counter takes less than a minute per sample for a typical cell count and it is compatible with a wide variety of eukaryotic cells Continued on next page Thawing and Establishing GIBCO Human NSCs continued Thawing 1 Remove the cells from liquid nitrogen storage and Procedure immediately transfer the cells to a 37 C water bath to prevent crystal formation 2 Quickly thaw the vial of cells by swirling it in the 37 C water bath and removing it when the last bit of ice has melted typically 2 minutes Do not submerge the vial completely Do not thaw the cells for longer than 2 minutes 3 When thawed immediately transfer the cells into a 50 mL sterile tube and carefully add 4 mL of pre warmed complete StemPro NSC SFM dropwis
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