Basic questions - (Dec/18/2014 )

Dear Guys,

I urgently need your help

In our lab we have an old model of BD facscallibur, (http://www.rpciflow.org/pdfs/manuals/facscalibur/FACS%20Calibur%20Instrument%20Guide.PDF)

So please do you have any advice, manual or experience regarding how to really optimize the electronic setting before acquisition on this machine.

Another question, if I am using only one cell type, does gating is important.

After fixation and permeabilization of my cells, where should be my cells on FSC/SSC graph (Only one type of cells).

if I want to study membrane bound protein like N-cadherin, shall I do fixation and permeabilization.

if I want to study different proteins in the same cells ( and I have only green 2ry AB ) so I must divide the sample and stain separately with different 1ry AB then stain with the same 2ry AB and observe each sample alone, shall my FSC/SSC should be the same for these samples (the same cells the same experiment, but testing protein is different, so FL-1 should be different).

I am sorry if this is a basic questions, but I really would appreciate your help

Madelin

Gating is important also with one cell type because you need to gate out debris and, if possible, doublets.

I worked with FACSCalibur, I don't really have any general advice but maybe if you have a particular problem...

Proteins where the epitope that is recognized by the AB is intracellular call for fixation/permeabilization. Surface proteins with extracellular epitope don't need it.

In your scenario, of course FSC and SSC should be the same in both samples - they are the same cells - if they got the same treatment regarding fixation etc. If FL-1 is for the green fluorescence then yes, it should be the only difference.

Hope it helps...

Thanks guys for your help :), It is really helpful