Cell Culturing Dr. Franco Lab

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Cell culture protocol (Franco Lab) Important notes: 1. Always wear gloves and lab coat while working with cells 2. Spray ethanol on the gloves before you will contact culture wares 3. After warming up and before opening up, the bottles for medium, PBS, antibiotics etc. should be sprayed with ethanol 4. Before and after using the biosafety hood, spray ethanol, and especially spray disinfectant after working with virus 5. Always use filtered pipet tips for cells 6. Avoid creating aerosols, especially while transferring the cells 7. Try to use long serological pipets or glass transfer pipets while aspirating medium from culture flasks or 15/50ml centrifuge tubes, or adding medium into, or resuspensing the cells 8. Your own clothes sleeve should be shorter than lab coat 9. Cells/virus/medium contaminated pipet tips or pipets or culture wares go to the biohazard waste bags, and non-contaminated waste go to general trash can 10. Tape the culture wares bag after using it 11. Do not warm trypsin, leave it at room temperature for 20min; warm medium no more than 20min

Medium: Base medium DMEM : F12 medium supplement with 10% fetal bovine serum and 1% antibiotics (penicillin-streptomycin (10,000units/ml) and 1% glutamine 2mM final concentration (200mM stock in -20oC, Invitrogen, cat#25030) 5% CO2 and 37oC incubator

Initial growth from frozen vial: 1. Take one vial of frozen cells from liquid nitrogen tank, thaw it in 37oC and transfer the cells into 10ml pre-warmed medium in a 15ml centrifuge tube before totally thawed. DO NOT ALLOW VIAL TO REMAIN THWAWED FOR LONG PERIODS OF TIME 2. Spin at 1300rpm x 5min, room temperature 3. Discard the supernatant and resuspend cells in 1ml medium with gently pipet up and down for 4-6 times 4. Then transfer cells to a T25 flask with 10ml complete medium 5. Change medium every 2 to 3 days 6. Wait and see until 80% confluence, then trypsinize and split

Splitting 7. Wash cells twice with PBS, then remove 8. Add 1x trypsin-EDTA (0.25% trypsin-0.53mM EDTA) diluted 1:1 with PBS and incubate at 37oC for 3-5min until detach. DO NOT LEAVE CELLS WITH TRYPSIN AFTER THEY HAVE BEEN DETACHED OR OVER 5 MIN 9. Add two times the amount of trypsin in fresh medium to neutralize trypsin and gently pipet up and down cells several times 10. Transfer cells to a centrifuge tube, and spin down at 1300rpm x 5min 11. Discard medium, add 1ml fresh medium and resuspend cells using p1000 pipette. Determine cell density and seed according to the table below. 12. Incubate at 37oC culture

Freezing cells 13. Trypsinize cells as above and determine cell density 14. Resuspend cells in 10% DMSO 90% fresh medium to adensity of 2 x 106 cells/ml. 15. Aliquot 1ml to each cryogenic vial, put in the freeze box (with isopropanol), -80oC for 24hours, then transfer to liquid nitrogen tank for long storage

Counting cells (with trypan blue exclusion) 16. After resuspending cells take 20ul cells and transfer them to a clean 0.5ml tube, mix with 20ul trypan blue (dilution factor) 17. Take 10-20ul mixture, and add from the gap between cover glass and hemocytometer chamber, until the cells fill the counting area. AVOID BUBBLES 18. Count 4 big 16-well squares, get a total number A, then cell density will be: (A/4) x dilution factor =***x104 cells/ml Cellculturecount.jpg Cellcultureguide.jpg

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