Dear colleagues, we are interested to study binding of a protein molecule on endothelial cells. The cells are treated with bacterial supe, which cleaves off receptors on endothelial cells. The binding protein is APC labelled and we use flow-cytometry.
Problem: The endothelial cells are treated with supernatent from bacteria. We can see a gradual decrease in binding of the protein (as expected), but the signal is slightly higher than negative control (no treatment with supe, unexpected). We think this might be due to the unspecific binding of the protein label with interfering factors in the bacterial supernatent.
We are interested in using the concentrated supernatent for this study, and not the purified proteins. Also CO-IP is not possible since elimination of a single factor is not expected to make things better.
My question is whether we can, after treatment with the supe fix our cell samples (with PFA), and afterwords incubate with labelled protein? Does anyone has a protocol or experience dealing with a similar problem?
Hi Shahan,
I am not sure what exactly you are doing and what is your method of fixing the cells. But in general after fixing the cells you basically denature most of intracellular structures and most likely you will not be able to do any biochemical assay with the fixed cell. Not 100% sure though!
Dear Shahan,
I think that fixing your cells with PF is not going to solve your problem, but will make it worse. As Shahid already noticed, you will crosslink your proteins, resulting in a reduced specificity for binding interactions. PF fixation is also known to increase autofluorescence of the cells, which will complicate your FCM analysis considerably. Have you tested if it is possible to do the binding of the ACS labelled protein before the bacterial supernatant treatment and measure the cells during the supernatant treatment? I understand from your question that you want to study the decrease of receptors for your ACS labelled protein as a function of bacterial super treatment. Is this correct?
I hope this may be helpful to you. Kind regards, Harrie Verhoeven