Cell washing - Can I use Tween/Triton etc? (Jun/29/2007 )
Hello,
I'm looking for a little advice. We are currently using a cell line transfected with a GFP reporter gene for DNA damage. We collect data spectrophotometrically and via flow cytometery. We have noticed that when we treat the cells with promutagens some compounds are metabolised to produce what appears to be adherent autofluorescent entities. I'm fairly confident that the metabolites aren't being internalised and are being retained on the cell surface.
My question is simply this: is it possible to wash live cells with a detergent and retain viability? (We currently measure viability with propidium iodide (on the flow) and to puncture the cells would give us false positive data.)
Any other advice as to how we could remove "sticky" metabolites from the cell surface before quantification would be appreciated.
Thanks in advance,
j
I'm looking for a little advice. We are currently using a cell line transfected with a GFP reporter gene for DNA damage. We collect data spectrophotometrically and via flow cytometery. We have noticed that when we treat the cells with promutagens some compounds are metabolised to produce what appears to be adherent autofluorescent entities. I'm fairly confident that the metabolites aren't being internalised and are being retained on the cell surface.
My question is simply this: is it possible to wash live cells with a detergent and retain viability? (We currently measure viability with propidium iodide (on the flow) and to puncture the cells would give us false positive data.)
Any other advice as to how we could remove "sticky" metabolites from the cell surface before quantification would be appreciated.
Thanks in advance,
j
the question is - if your idea is right - how interact the metabolites with the plasma membrane? with glycocalyx? protein ectodomains? lipids? steroids? isn´t there an enzyme which will lyse/digest your metabolit but would not harm the cells?
detergents may harm the cells, and give unspecific stimulation of cells; moreover, one has to check various detergents, mixtures of detergents and their concentrations...
Using Triton or Tween would not be a good idea, since they solubilize the membranes. You may try to wash extensively with PBS. Using something stronger may harm your cells and again give you false positive.
Try a mild trypsin treatment see if you can remove these from your cells.
triton or tween? err... i use them to lyse my cell in my infection assay...
Acid wash (using low pH buffer e.g. acid acetic) or high salt (High concentration of NaCl) or combination of both may help to wash away the metabolites that bind to cells. That is what we use when we study endocytosis in live cells and want to wash away the uninternalized ligand that binds to receptor, e.g. EGF, transferrin... There are many protocols for these, check in PubMed and try using mild conditions first before progress to harsher ones.
i wash my cell with 0.9% NaCl
many thanks for all the helpful comments. i think i have enough food for thought there.
j