pp 314, Ian Freshney
Materials:
Sterile:
Supernatant medium free of antibiotics from 7-day monolayer or centrifuged suspension cell culture
Indicator cells 3T6, NRK, Vero, A459
Petri dishes
Non sterile:
Hoechst 33258 stain, 50ng/ml in PBS without phenol red (BSS-PR) or D-PBSA
BSS-PR: Hank’s BSS without phenol red
D-PBSA – Dulbecco’s PBS without Ca+2 and Mg+2
Fixative: freshly prepared acetic methanol (1:3 on ice)
Buffered glycerol moutant
Protocol:
Check index for location of ampoules to be thawed
Collect all materials, prepare medium and label culture flask
Retrieve ampoule from freezer, check from label that is correct one and if it has not been submerged in liquid nitrogen, place it in sterile water at 37oC in a beaker, plastic bucket/water bath. If possible, avoid getting water up to the cap as this will increase chance of contamination
When ampoule has thawed, double check label to confirm identity of cells, then swab ampoule thoroughly with 70% alcohol, and open it in LAF
Transfer contents of ampoule to a culture flask with 1-ml piepette
Add medium slowly to cell suspension: 10 ml over about 2 min added drop-wise at the start, and then a little faster, gradually diluting cells and cryoprotectant
For cells that require centrifugation to remove cryoprotectant :
Dilute cells slowly as per step 6 but in a centrifuge tube or universal container
Centrifuge them for 2 min at 100g
Discard supernatant medium with cryprotectant
Resuspend cells in fresh growth medium
Seed flsk for culture
The dregs in ampoule may be stained with Napthalene Black or Trypan Blue to determine their viability
Check after 24 hrs
For attached monolayer cells, confirm attachment and try to estimate percentage survival based on photographs of cells at the expected density (cells/cm2) with full survival For suspension growing cells check appearance (Clear cytoplasm, lack of granularity), and dilute to regular seeding concentration. This can be made more precise if the cells are counted and an estimate of viability is made, in which case the cells can be diluted to regular seeding conc.
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