Protocol 2:
Remove medium
Wash in PBS to remove any serum
Add 5 ml trypsin; let sit 5-10'
Bang cells off flask.
Add 5 ml serum medium to inactivate trypsin and wash remaining cells off bottom of flask.
Spin down cells at 1200-1400 rpm for 5 min.
Resuspend cells in serum medium.
Aliquot cells into new flasks accordingly

Protocol 3:
Detach cells from the flask either by banging or scraping
Pipet cells up and down about 10 times to resuspend cells and separate clumps
Aliquot cells into new flasks accordingly

BG2

Remove medium (save and filter through syringe filter for use as "conditioned medium").
Wash in PBS to remove any serum
Add 5 ml trypsin; let sit 5-10'
Bang cells off flask.
Add 5 ml serum medium to inactivate trypsin and wash remaining cells off bottom of flask.
Spin down cells at 1200-1400 rpm for 5 min.
Resuspend cells in conditioned medium.
Aliquot cells into new flasks accordingly

Can also use Accutase instead of trypsin.

IV. FREEZING

Grow cells to subconfluencey - approximately 1-2 x 10⁷ cells/ml.
Label appropriate # of cryovials.
Remove cells from flask (trypsinize and resuspend in medium if necessary).
Count cells.
Spin cells at 1200 rpm 5'
Aspirate off medium.
Resuspend at approximately 1.1 x 10⁷ cells/ml in freezing medium:
  FBS + 10% DMSO, filtered OR Complete medium + 10% DMSO, filtered
Aliquot 1ml cell suspension/cryovial.
Put vials in foam box, tape closed, and place in -70^oC. After a few days, transfer vials to LN₂ for long term storage.

V. THAWING

Prepare 15ml conical tube with 5ml medium.
Thaw cells quickly in water bath.
Just before cells are completely thawed, decontamiate the outside of the tube with 70% EtOH.
Transfer the cells to the conical tube with 5ml medium.
Spin at 1200rpm 5'.
Aspirate and resuspend cells in 5ml medium.
Plate in a T25 flask. Watch daily. Initially, cells may need to be split at irregular intervals.

VI. COUNTING

Remove ~0.5 ml cells from flask (trypsinize cells if necessary) into eppendorf.
Dilute cells into Trypan Blue according to estimated density.
(confluent cultures try 30:70 µl to 50:50 µl of cells:TB).
Transfer 10 - 12 µl cells to haemocytometer.
Count cells in opposite corners (~100-200/ large square).
Calcuation:
    (count #1 + count #2) / 2 x (dilution factor) x 10⁴ = X cells/ml (usually ~1-10 x 10⁷)

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