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crystal violet adhesion assay and serum - should serum in media be excluded (May/19/2008 )

hi all,

i think that perhaps i have been in the lab for too long today and can't get my head to work anymore. so, i would like to do a cell adhesion assay where i let cells adhere to a substrate and then rinse after some time, fix, stain with crystal violet and then solubilize and read absorbance.

my question is this: should i use serum-free media when i seed cells? is it alright to use the culture media (with serum) because to show how the cells adhere to the substrate? if i use serum-free media, is it also necessary to serum starve the cells? i guess i'm sort of confused as to how much serum masks the adhesion process (i've been under the assumption that it is needed to assist in adhesion).



It depends- are you trying to look at the influence of serum on the cell attachment? Do you normally grow your cells with serum?

I would say, if you normally grow your cells with serum, and you aren't trying to look at serum's influence on the cells, then you should grow them with serum as this is the normal condition for the cells. I don't think the serum will mask anything that can be shown by crystal violet, unless the attachment factors are found in serum at high concentration.
Serum staving the cells before seeding is probably not a good idea, it forces the cells into G1 arrest and they may not attach from this stage (anyone who knows more about this feel free to correct me).

Just remember to wash the cells well with PBS before adding the crystal violet as the serum will also stain.



I am doing dayly CV. I always use a media with serum and I never had any problem.



One more question: how important is the temp and CO2? I know that 5% and higher CO2 will encourage cell adhesion, but I left my cells in the hood for 30 min to attach in an adhesion assay. If I test all of my conditions at RT under the same conditions, will this be a problem?