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Cell proliferation assay for LNCaP cells - (Jan/06/2010 )

Hi,

I need to do cell proliferation assay for LNCaP cells after stimulation with androgen. The protocol I follow is I grow the cells in 96 well plate in 10% complete medium for 48 hrs. then remove the medium, give a PBS rinse of the plate and then add medium with 2% charcoal stripped serum. After adding medium I stimulate with different doses of androgen. However, the problem is, since LNCaP cells are loosely adherent cells, they tend to come out when I aspirate medium or rinse with PBS. They also come out when I add fresh medium with 2% CS FBS. As a result, the cell number varies.. which in turn will give me a wrong cell proliferation index. IF somebody cud help me on this topic..

-sheebs-

try coating the plate with a cell attachment factor such as collagen (gelatin) or fibronectin.

-bob1-

i usually use polylysine to precoat the plate, and this maybe help some.

http://www.genlantis.com/objects/catalog/p...%20Protocol.pdf

-shuiyi-

sheebs on Thu Jan 7 07:42:13 2010 said:


Hi,

I need to do cell proliferation assay for LNCaP cells after stimulation with androgen. The protocol I follow is I grow the cells in 96 well plate in 10% complete medium for 48 hrs. then remove the medium, give a PBS rinse of the plate and then add medium with 2% charcoal stripped serum. After adding medium I stimulate with different doses of androgen. However, the problem is, since LNCaP cells are loosely adherent cells, they tend to come out when I aspirate medium or rinse with PBS. They also come out when I add fresh medium with 2% CS FBS. As a result, the cell number varies.. which in turn will give me a wrong cell proliferation index. IF somebody cud help me on this topic..

Hi, Sheebs, have you solved your problem about lncap cells? I am doing experiments using lncap cells too, I faced the same problem as you, but I did it very carefully, the variation seems OK, but the effect of DHT is not so significant after 72hrs treatment(I use DHT as a postive control), how you did your experiment? what's your effect looks like? maybe we can exchange our opinions on lncap cells, what do you think?

-isabellaweng-

years later and I have the same questions...did you succeed? :)

-liezl-