Neural Progenitor User Manual
Contents
1. Neural Progenitor User Manual AFUNA BIOMEDICAL TABLE OF CONTENTS Page OCO e EE 2 Maters REGU e EE 3 Media Storage and Handling 00 4 Preparation of Poly Ornithine and Laminin Coated Dlates eee cece eee 5 awe Neral Progenitor CCS EE 6 Subculture of Neural Progenitor Cell 7 ee Eh Wl EE 8 EE 9 EE CS 10 Staining for Intra cellular Proteins cece cece ence eee eneeeeneeeeenaeeeseeaeenaeenes 11 Staining for Extra cellular Proteins c cece cece cece ee nc eee eeeseeeeeeseeeseeaeenaeeees 14 Appendix heath oes E 16 RE E og eee 17 KEE Ke ee eee eee 18 To learn more about Aruna Biomedical Inc visit our website at www arunabiomedical com ArunA Biomedical Inc www arunabiomedical com INTRODUCTION ENStem A Human Neural Progenitor Cells Aruna Biomedical has developed a method to derive Neural Progenitors from NIH registered human embryonic stem cells hESCs This allows us to produce user friendly turn key kits containing cryopreserved Neural Progenitors and all the reagents needed to propagate and differentiate Neural Progenitors into primary cultures of neural cells The technical innovation is that our Neural Progenitors and their derivatives will proliferate as adherent monolayer cell cultures As a direct result of this advance our Neural Progenitor cell products can be reliably differentiated for quantitative studies2. Prepare primary antibody 1 Ab in 1 ml of BLOCKING SOLUTION at the recommended dilution Aspirate BLOCKING SOLUTION out of slide wells Add enough 1 Ab solution made in step 5 to completely cover the cells in each well 250 ul well Cover and incubate 1 hour at room temperature This can be extended to over night at 4 C if necessary Wash wells 4 times in HIGH SALT BUFFER for 5 minutes each wash Prepare secondary antibody 2 Ab in 1 ml of BLOCKING SOLUTION at recommended dilution while completing washes in step 8 Aspirate last wash ArunA Biomedical Inc 12 www arunabiomedical com 11 12 13 14 15 16 17 18 19 20 21 22 23 Add enough 2 Ab solution made in step 9 to completely cover the cells in each well 250 ul well Incubate 1 hour at room temperature During incubation protect sample from light to prevent fluorescence bleaching Wash cells 3 times in PBS While completing washes in step 10 prepare DAPI SOLUTION ata 1 10 000 dilution in distilled H20 Add enough DAPI to solution to completely cover the cells in each well 250 ul well Incubate for 5 minutes at room temperature Protect from light during incubation Wash cells 3 times in PBS Aspirate off PBS Gently remove sides of chamber Tilt slide to one side and allow excess PBS to run off onto paper towel Place one drop of mounting media directly in center of each well area We recommend INVITROGE
3. Remove excess mounting media from slide and cure in the dark for 24 hours Seal with nail polish on all four sides Keep in dark storage until results are observed and documented ArunA Biomedical Inc 15 www arunabiomedical com CHARACTERIZATION OF ENStem A HUMAN NEURAL PROGENITOR CELLS cells F e Ge d E Ce Ze 2 17 2e AA PE CS ui Vo PE nestin SOx 2 i ri ba ei th ey Zi Figure 1 ENStem A Human Neural Progenitor Cells Part No SCCO03 are grown as monolayers A are karyotypically normal B and express NSC markers Nestin C D green Part No MAB5326 1 500 and Sox 2 C E green Part No AB5603 1 1000 Nuclei of the cells were visualized with DAPI blue The Sox 2 transcription factor is co localized with the DAPI blue staining in the nucleus F ArunA Biomedical Inc 16 www arunabiomedical com Figure 2 ENStem A Human Neural Progenitor Cells Part No SCCOO3 are negative for Oct 4 staining A B while control H9 human embryonic stem cells are positive for Oct 4 staining C Part No MAB4401 Mouse feeder cells are also negative for Oct 4 staining arrows ArunA Biomedical Inc 17 www arunabiomedical com Figure 3 ENStem A Human Neural Progenitor Cells can differentiate into multiple neuronal subtypes Using ENStem A Neuronal Differentiation Medium Part No SCMO17 a majority of the cells exhibit a neuronal phenotype A HI tubulin red Part No M
4. by pipetting up and down to manually detach them from the plate Cells should be triturated into an almost single cell suspension prior to passaging We recommend using a 200 ul or 1000 ul manual pipette for 1 2 minutes depending on size of plate to detach the cells Alternatively cells can be detached with a sterile cell scraper and then triturated into a single cell suspension Plates should be observed to ensure that all cells have been removed This is most easily accomplished by working under a dissection microscope within a biosafety cabinet but can also be achieved by frequent observation under bright field or phase contrast microscopes If necessary cells can be centrifuged at 200 x g for 4 minutes in order to resuspend the pellet at the necessary concentration Cells can now be plated at the appropriate density onto a coated plate or frozen We recommend culturing the cells at a high density by passaging 1 2 1 3 Replace the media with fresh pre warmed NEURAL EXPANSION MEDIUM 24 hours later and every other day therafter ArunA Biomedical Inc 7 www arunabiomedical com CHARACTERIZATION Every lot of Aruna Biomedical Neural Progenitor cells has been tested for high expression levels of Nestin and Sox 2 and low expression levels of Oct 4 ENSTem A Neural Progenitor Cells have the ability to differentiate into multiple neuronal phenotypes and maintain a normal karyotype after multiple passages Cells have also bee
5. AB1637 1 1000 Cultures contain some GABAergic C GABA red cells that express the transcription factor HB9 D Part No AB5963 1 50 and also cholinergic cells E ChAT green Part No AB144P 1 100 GABAergic cells colabel with the neuronal marker I I tubulin B D Cell nuclei were visualized with DAPI blue ArunA Biomedical Inc www arunabiomedical com 18
6. N S PROLONG GOLD At an angle gently lower a cover slip onto the slide trying to avoid air bubbles where possible Remove excess mounting media from slide and cure in the dark for 24 hours Seal with nail varnish on all four sides Keep in dark storage until results are observed and documented ArunA Biomedical Inc 13 www arunabiomedical com STAINING FOR EXTRA CELLULAR PROTEINS SOLUTION PREPARATION BLOCKING SOLUTION Catalog Amount ee PDS Chemicon BSS 1005 B 9 4 ml with Catt and Mer l 6 serum from the animal that your 600 ul secondary antibody is produced in H e Mix by inversion Make fresh Blocking Solution for each use e Used in steps 3 4 5 and 8 STAINING FOR EXTRA CELLULAR PROTEINS Pre made solutions you will need Catalog Required PBS Steps with Car and Mer E E 2 12 and 15 DAPI VWR 80051 386 Steps 0 and 14 Prolong Gold P36930 Step 19 1 Fix cells according to protocol All manipulations should be carried out with extreme care as cells may dislodge easily We recommend adding and removing liquids slowly with a manual pipetteman or transfer pipette 2 Wash wells 2 times in PBS with Mg and Ca PBS Note For all washes wells should be completely filled with solution 3 Add enough BLOCKING SOLUTION to completely cover the cells in each well we recommend 250 ul well Incubate at room temperature for at least 45 minutes 4 Prepare primary antibody 1 Ab in 1 ml of BLOCKING SOLUTIO
7. N at the recommended dilution ArunA Biomedical Inc 14 www arunabiomedical com 11 12 13 14 15 16 17 18 19 20 21 22 Aspirate BLOCKING SOLUTION out of slide wells Add enough 1 Ab solution made in step 4 to completely cover the cells in each well 250 ul well Cover and incubate 1 hour at room temperature This can be extended to over night at 2 8 C if necessary Wash wells 4 times in PBS for 5 minutes each wash Prepare secondary antibody 2 Ab in 1 ml of BLOCKING SOLUTION at recommended dilution while completing washes in step 7 Aspirate off last wash Add enough 2 Ab solution made in step 8 to completely cover the cells in each well 250 ul well Incubate 1 hour at room temperature During incubation protect sample from light to prevent fluorescence bleaching Wash cells 3 times with PBS While completing washes in step 11 prepare DAPI SOLUTION ata 1 10 000 dilution in distilled H2O Add 250 ul of DAPI SOLUTION to each well Incubate for 5 minutes at room temperature Cover with foil during incubation Wash cells 3 times in PBS Aspirate off PBS Gently remove sides of chamber Tilt slide to one side and allow excess PBS to run off onto paper towel Place one drop of mounting media directly in center of each well area We recommend INVITROGEN S PROLONG GOLD At an angle gently lower a cover slip onto the slide trying to avoid air bubbles where possible
8. dding media all at once may result in osmotic shock Centrifuge at room temperature at 200 x g for 4 minutes Carefully aspirate as much of the supernatant as possible Note Steps 4 6 are very important in the removal of residual DMSO Resuspend the cells in 2 ml of NEURAL EXPANSION MEDIUM Plate the cell suspension at a density of 1 vial Approximately 1 x 106 cells per 35 mm poly ornithine and laminin coated tissue culture dish Incubate the cells at 37 C in a 5 CO2 humidified incubator After the cells have incubated for 24 hours the NEURAL EXPANSION MEDIUM in the plate should be aspirated and replaced with fresh medium Medium should be changed every other day thereafter Once the Neural Progenitor cells reach 90 100 confluence they can be dissociated manually and passaged or alternatively frozen for later use The cells should be maintained at a high density at all times and passaged at a 1 2 or 1 3 dilution ArunA Biomedical Inc 6 www arunabiomedical com SUBCULTURE OF NEURAL PROGENITOR CELLS Note This process does not utilize enzymatic digestion 1 Once the Neural Progenitor cells reach 90 100 confluence carefully remove the media from the poly ornithine and laminin coated 35 mm tissue culture plate containing the Neural Progenitor cells Add pre warmed NEURAL EXPANSION MEDIUM to the plate 2 m1l 35 mm 7 ml 100 mm so that cells can be harvested in fresh medium The cells should then be manually passaged
9. hesion and growth 1 Thaw the poly ornithine and laminin slowly at 4 C Dilute poly ornithine to 20ug ml in cold sterile ddi water Dispense poly ornithine solution to completely cover the bottom of the dish 2 m1 35 mm 5 ml 100 mm Incubate in a humidified 37 C incubator for at least 1 hour Remove the poly ornithine solution and rinse once with sterile ddi water Dilute laminin to Sug ml in cold sterile ddi water Dispense laminin solution to completely cover the bottom of the dish 2 ml 35 mm 5 ml 100mm Incubate in a humidified 37 C incubator for at least 1 hour Transfer plates to 2 8 C and store plates in laminin solution for up to 3 weeks Store at 2 8 C for up to 3 weeks To use bring up to room temperature leave on bench top or place on warm plate set at 37 C Aspirate the laminin solution and rinse one time with PBS before use ArunA Biomedical Inc 5 www arunabiomedical com 10 11 12 THAWING NEURAL PROGENITOR CELLS All materials should be ready prior to thawing vial Cells are thawed rapidly by hand rotating the cryovial in a 37 C water bath Note Thaw rapidly for maximum cell viability Spray vial with 70 ethanol and dry before placing in a biological safety cabinet Working in a biological safety cabinet transfer the thawed cells into a sterile 15 ml conical centrifuge tube Swirling slowly drop wise add 10 ml of pre warmed NEURAL EXPANSION MEDIUM to the cell suspension Warning A
10. ical Inc 9 www arunabiomedical com FIXING CELLS WITH PARAFORMALDEHYDE SOLUTION PREPARATION 2 PARAFORMALDEHYDE PFA Catalog Amount per Final J T B Paraformaldehyde Baker S898 07 2 grams PBS no ua Chemicon ESS 10055 100m TZ Additional supplies include Whatman filter paper 42 Cat 1442125 Note Paraformaldehyde is toxic see MSDS 1 Heat 75 ml of PBS to 56 C 2 Add 2 grams of paraformaldehyde to the solution Add 2 drops of 1M NaOH from a transfer pipette 3 Once in solution use pH meter or pH paper and adjust the pH using HCl or NaOH to 7 4 4 Filter using Whatman filter paper Using PBS adjust volume up to 100 ml D Store at 2 8 C and use within 1 week FIXING CELLS 1 Carefully remove spent media with a pipetteman or transfer pipette being careful not to dislodge cells Wash cells 1 time in PBS with Ca and Mg PBS Working in a fume hood add PFA solution to cover the bottom of slide wells 250 uL well 4 Incubate at room temperature for 15 20 min Wash cells 3 times with PBS Store fixed cells with PBS at 2 8 C until ready to stain We recommend staining as soon as possible after fixation preferably in the same day ArunA Biomedical Inc 10 www arunabiomedical com STAINING FOR INTRA CELLULAR PROTEINS SOLUTION PREPARATION HIGH SALT BUFFER Reagent Vendor Catalog Amount Final Number per L Concentration Sodium Chloride S7653 14 61 g 250mM e Mix by inver
11. n confirmed to be negative for mycoplasma Please refer to figures 1 2 and 3 for sample images ArunA Biomedical Inc www arunabiomedical com DIFFERENTIATION The differentiation protocol media provided is intended as a basal system For specific cell types differentiation protocols including additional neurotropic factors can be developed as determined by the end user s interests Note There may be considerable cell death throughout this procedure 1 Begin the differentiation process by first removing the NEURAL EXPANSION MEDIUM 2 Wash the plate with PBS containing Catt and Mg 3 Add pre warmed NEURONAL DIFFERENTIATION MEDIUM to the plate 2 ml 35 mm 7 ml 100 mm 4 Incubate in a humidified 37 C incubator with 5 COs 5 After 2 to 3 days when the plate is confluent passage the plate 1 2 in NEURONAL DIFFERENTIATION MEDIUM following the subculturing protocol 6 Incubate in a humidified 37 C incubator with 5 CO2 Replace media with fresh pre warmed NEURONAL DIFFERENTIATION MEDIUM every 3 4 days After the desired differentiation period cells will begin to increase expression of the neuronal marker B tubulin within 1 week and will have a high percentage of B tubulin expression by the second week cells can be harvested Neuronal Differentiation Medium and replated on a poly ornithine and laminin coated chamber slide we recommend 50 000 150 000 cells 300 ul well for staining ArunA Biomed
12. o P3655 ANNE E Sigma Part No L2020 EE Millipore Part No TMS 002 C PENICILLIN STREPTOMYCIN SOLUTION Millipore Part No TMS AB2 C PHOSPHATE BUFFERED SALINE 1X PBS Millipore Part No BSS 1005 B ArunA Biomedical Inc 3 www arunabiomedical com MEDIA STORAGE AND HANDLING For thawing and subculture of cells ENStem A Human Neural Expansion Medium ENStem A Neural Expansion Medium should be stored at 20 C until ready to use Upon thawing fresh L Glutamine should be added to the ENStem A Neural Expansion Medium for a final concentration of 2 mM FGF 2 should be added fresh to the ENStem ATM Neural Expansion Medium for a final concentration of 20ng ml before each use Thawed medium should be stored at 2 8 C and given a 1 month expiration date Protect from light and wide swings in temperature Basic FGF 2 50 ug lyophilized should be reconstituted with 100 uL 5 mM Tris HCL pH 7 6 for a final concentration of 100 ug ml Dispense into aliquots to avoid repeated thawing Store at 20 C COMPLETE NEURAL EXPANSION MEDIUM Ras Amount Amount 6 per ml per 100 ml ATM Expansion Medium ge i ArunA Biomedical Inc 4 www arunabiomedical com PREPARATION OF POLY ORNITHINE AND LAMININ COATED PLATES We recommend using poly ornithine and laminin coated plates to culture Neural Progenitor cells Poly ornithine and laminin coated plates provide the optimal foundation for ad
13. of neurons and glial cells Aruna s Neural Progenitor cell products will benefit biomedical research in both neurological disorders and the basic science of human developmental pathways Thanks to Aruna Biomedical more researchers will have ready access to adherent monolayer human neural cells allowing for quantitative analysis for either small scale or high throughput content screens We look forward to this new technology leading to the discovery of novel therapeutic compounds tests for neurotoxicity and breakthroughs in understanding human neural development We also look forward to hearing your feedback on our product For support and technical assistance contact us at info arunabiomedical com techsupport arunabiomedical com For Research Use Only not for use in therapeutics or diagnostics ArunA Biomedical Inc 2 www arunabiomedical com MATERIALS REQUIRED ENStem A HUMAN NEURAL PROGENITOR CELLS Chemicon Part No SCCO03 http www millipore com catalogue item scr0O55 ENStem A NEURAL EXPANSION MEDIUM Chemicon Part No SCM004 http www millipore com catalogue item scm004 ENStem A NEURONAL DIFFERENTIATION MEDIUM Chemicon Part No SCMO17 http www millipore com catalogue item scm017 ENStem A Neural Freezing Medum Chemicon Part No SCM0O11 http www millipore com catalogue item scm011 POLY L ORNITHINE HYDROBROMIDE users eee Sigma Part N
14. sion until completely in solution Store at room temperature for up to 2 months e Used to make Blocking Solution and Permeabilization Buffer as well as in step 8 BLOCKING SOLUTION EE Amount 8 per 10 ml High salt buffer 9 4 ml 6 serum from the animal that your 600 ul secondary antibody is produced in g e Mix by inversion Make fresh Blocking Solution for each use e Used in steps 4 5 6 and 9 PERMEABILIZATION BUFFER Catalog Amount coe EMD e Mix by inversion until completely in solution Store at room temperature for no longer than 2 months e Used in step 3 ArunA Biomedical Inc 11 www arunabiomedical com STAINING FOR INTRA CELLULAR PROTEINS Pre made solutions you will need Catalog Required PBS Steps with Ca and Mer EE 2 12and15 DAPI VWR 80051 386 one and 14 Prolong Gold P36930 Step 19 Fix cells according to protocol All manipulations should be carried out with extreme care as cells may dislodge easily We recommend adding and removing liquids slowly with a manual pipetteman or transfer pipette Wash wells 2 times in Catt and Mg Note For all washes wells should be completely filled with solution Wash with PERMEABLIZATION BUFFER 3 times for 5 minutes each In this step you are permeablizing your cells so time is very important Add enough BLOCKING SOLUTION to completely cover the cells in each well we recommend 250 ul well Incubate at room temperature for at least 45 minutes
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