Reconstitution, propagating and freezing cell lines

Materials

  1. Culture medium (usually DMEM+10%FBS+1%PS unless otherwise indicated)
    • Thaw FBS (Hyclone SH30088.03) and PS (short for Penicillin-Streptomycin Solution, 100X, Mediatech 30-002-CI ) in 4oC refrigerate overnight.
    • In Larminar Flow Hood, to 500ml DMEM ( Mediatech 10-013-CV ) , add 56.2ml FBS and 5.6ml Pen-Strep, mix well. Store at 4oC. Pre-warm at RT before use.
  2. Freezing medium: Culture medium containing 7.5%DMSO
  3. Trypsin EDTA (Mediatech 25-051-CI)
  4. Sterile PBS solution ( Mediatech 21-031-CV)
  5. T75 tissue culture flasks with 0.2u vent caps (or dishes)
  6. 1.2 or 2ml cryogenic vials
  7. Nalgene® Cryo Freezing Container (filled with isopropanol)
  8. Liquid nitrogen storage container.
  9. Serological pipettes
  10. 50ml sterile tubes
  11. 37oC water both
  12. Deposable sterile Pasteur pipettes
  13. 37oC incubator with 5% CO2
  14. Larminar flow hood with vacuum adapter
  15. Centrifuge with 50ml tube adapters
  16. 70% ethanol
  17. Wipe papers
  18. Marker pen
  19. Safety Goggles are recommended when taking frozen cells from liquid nitrogen
Procedure
Cell reconstitution
  1. Pre-warm medium to room temperature
  2. Take a vial of cells from liquid nitrogen storage tank, and quickly thaw the vial on a 37oC water bath.
  3. Wipe the vial with 70% ethanol and let dry in hood.
  4. Uncap the vial, transfer the cell suspension into a T75 flask. Discard the empty vial in a biohazard container.
  5. Using a new pipette, add 12ml culture medium to the flask and pipet up and down a couple of times to break cell clumps and mix the suspension.
  6. Put the flask in incubator and replace fresh medium the next day.
  7. Change medium twice a week until the cells reach subconfluent.
  8. When reach subconfluent, the cells can be used for experiment, subcultured or freezed for futhure usage.

Cell propagation

  1. Aspirate the medium in the flask with Pasteur pipette.
  2. Wash cells -2 times by adding 12ml sterile PBS and aspirating off.
  3. Add 3.5ml trypsin-EDTA, shake to cover the whole cell surface. Incubate at 37oC for 3-10min depending on cell lines.
  4. Once the cells detach from the flask, neutralize the trypsin by rinsing cells with 7ml culture medium and pipet up and down to break cell clumps.
  5. Dilute cells with culture medium at 1:3-20 depend on cell line and purpose.
  6. Plate 12ml each in new flasks and incubate at 37oC with 5% CO2.

 

Readings:
Hosted by www.Geocities.ws

1