Sample prep of cells for FACS analysis and running WB - (Feb/17/2015 )

Hi there, 

Im not too knowledgable about sample prep for FACS so I was curious, 

Can the same cells used for prepping for FACS analysis be used for running WB?

I just transfected cells with my gene of interest, and then my colleague prepped them for FACS analysis, and then gave me the pellet to lyse afterwards. 

Is this appropriate?

Many thanks for advice. 

I don't see why there should be a problem, assuming that by prepping you just mean preparation of the cells for staining and not the actual staining procedure itself.

Sorry, I did not understand what you mean by staining. 

I transfected seeded cells, and then my colleague used the cells 24 hrs later by PBS washing the cells and putting them in a FACS tube and then running the assay.

She centrifuged and got the pellet and gave it to me to run WB.

Im just wondering how samples used  for FACS analysis (which from my understanding will suck up the sample), can be used afterwards to study protein expression. 

I think its possible but accurately judging protein expression....that sounds like another story

well its certainly possible to acquire and use cells used for FACS analysis in experiments afterwards; think of FACS sorting machines for example. Or maybe she gave you the rest which was not sucked up in case a simple FACS machine was used.

when i said staining i assumed your colleague was perhaps staining the cells for a certain molecule using an antibody. What exactly was done with the cells during the FACS assay, were they just measured "natively" for size and granularity ? Or was the fluorescence of a transfection reporter gene measured to ensure successful transfection rates ?

Its Bimolecular fluorescence complementation (BiFC). In my case, one plasmid has one part of a venus construct with the gene of interest, and then theres a second plasmid which has the other end of Venus with the same gene of interest. When they come together, such as when I transfect them both in the same well, they are supposed to light up, and then we can see that through FACS and also WB.  So we are measuring more the fluoresce of the reporter gene. 

My colleague PBS washed the cells into a FACS tube, and then gave me the cell pellet afterwards for me to lyse. 

Im not very clear as to how the pellet can go from a FACS tube to another to be WB analyzed....