flow cytometry - labelling surface receptor on monolayer cells (Jul/07/2006 )

hello members,
i have a small question about flow cytometry.
i want to lebel endothelial cell (adharent cells)surface receptor with FITC labelled antibody. in regard to this procedure, following r the steps to proceed.
1-trypsinize cells,
2-label cells with FITC-Ab
3-fix in parafarmaldehide
4-go for analysis

which of the following step in the method u prefer???

a--1234
b--2314
c--3214
d--3124

if you have any other ideas, please share with me.

thanks in advance.
payeli.

Honnestly I never did FACS on adherent cells, but I guess that trypsine might damage the surface receptor you want to label?

However, I would start by... trypsine (or scraping cells... let's see what the others will say), so it's easier to do the labeling, it's also a smaller volume, you will spare antibodies,
then , incubate with antibodies (PFA could modify epitopes for your antibody)
then fix with PFA
then go for analysis.

My answer is a--1234. "et c'est mon dernier mot Jean-Pierre". (sorry, joke for french people. it's in this TV game, who wants to be a millionnaire. when you answer and do not change your mind, you say c'est mon dernier mot. Jean Pierre is the first name of the french host)

What do I win?

I routinely do FACS on airway epithelial cells (adherent).

All labeling is done on the tissue culture plates.

1) Block 15 minutes 5% normal donkey serum in PBS
2) incubate with primary antibody 1 hour in 5%NDS/PBS
3) Wash 3x 5 min with PBS
4) incubate with secondary antibody 30 min in 5%NDS/PBS
5) wash 5x 5 min with PBS
6) Treat with 0.02% EGTA in Hanks Balanced Salt Solution w/out phenol red calcium or magnesium
after about 30 minutes the cells will lift off. You can scrape the rest with a pipette tip when you are transferring them into the next step.
7) Transfer cells to 2% Paraformaldehyde so that the final concentration will be 1%

This works well for proteins with extracellular epitopes. You can add saponin or Triton to permeabilize if you want to look at a cytosolic protein, but this makes the cells much more fragile and will be noticeable on your FSC v SSC plots.

I have done flow cytometry specifically to measure surface binding of a recombinant protein to epithelial cells and found that trypsinisation was out of the question due to degradation of the surface receptor- so i used alternative methods to lift the cells- i.e. mechanical scraping or EDTA incubation. This enabled binding. Perhaps this would be useful for you aswell since you are examining a surface protein?

For my protein, I had to avoid trypsin to get staining. I usually do liike Finnbar does. I use 5mM EDTA in HBSS without magnesium or calcium.

I think you should use a non-enzymatic cell detachment method using EDTA and cell dissociation solution to detach your ECs.