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how to dissociate cells for cell migration assay - (Aug/31/2010 )

Hallo,

I am planning to perform cell migration assays (MCF7) cells. My question is how to dissociate the cells before placing them in the upper chamber for migration? My problem is that if I use trypsin, it needs to be inactivated, but in my assay I need to work with cells that are starved (no FBS)

-naomi-

Use trypsin and soy-bean trypsin inhibitor... Sigma has it.

-bob1-

thanks, I will give it a try. I was wondering whether at all trypsin may effect cell migration as it may cleave proteins on the cell surface that effect the process?

-naomi-

yoe are right..
trypsin can cleave off some cell surface receptors , and u should check if your's does too..
if not, i guess it is fine..

i usually use the citric saline method to harvest my cells,
as the receptor im studying on gets cleaved by trypin..

Citric saline (from Lesley Barber, Melbourne, Australia)
(a) Less harsh than EDTA or trypsin, good viability.
(B) Recipe for 10X stock solution of citric saline
(i) 1.35 M potassium chloride (KCl, MW=74.55, 10.06 g/100 ml)
(ii) 0.15 M sodium citrate (C6H5Na3O72H2O, MW=294.1, 4.41 g/100 ml)
(iii) Autoclave and store at 4 C
(iv) Dilute 1:10 with sterile distilled water before use
(c) Protocol
(i) Remove all media, flood flask bottom with 1X citric saline
(pre-warmed to 37 C)
(ii) Incubate at 37 C for 4 minutes
(iii) Tap flask bottom to gently detach all cells

-raaann-