I urgently need your help
In our lab we have an old model of BD facscallibur, (http://www.rpciflow....ument Guide.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