Hi all,

As i am new to this forum first i'd like to say "hello" to everybody...

I am trying to evaluate a surface antigen (a CD one) on epithelial cells by FCM, but as i trypsinize cells to get a single cell suspension it has been realized that cells loose their surface proteins.
Then i tried to detach cells by scraping, but this caused clumps. Even i passed these cells through 70 um nylon mesh, cells appeared in non-homogenous populations at FS and SS. Then i tried EDTA-PBS mixture it worked better (but not good enough to maintain a single cell suspension) but i am worrying about if EDTA interferes with antibody binding.

Does anyone know a good protocol for flow cytometric analysis of surface antigens on adherent cells?

And i wonder if the doublets or triplets or bigger cell clumps may interfere with positive cell population and fluorescence intensity (FITC)?
Is it better to consider smaller fraction of population (~65%) composed of single cells for analysis?

Thanx...

gunes

gunes said:

Hi all, As i am new to this forum first i'd like to say "hello" to everybody... I am trying to evaluate a surface antigen (a CD one) on epithelial cells by FCM, but as i trypsinize cells to get a single cell suspension it has been realized that cells loose their surface proteins. Then i tried to detach cells by scraping, but this caused clumps. Even i passed these cells through 70 um nylon mesh, cells appeared in non-homogenous populations at FS and SS. Then i tried EDTA-PBS mixture it worked better (but not good enough to maintain a single cell suspension) but i am worrying about if EDTA interferes with antibody binding. Does anyone know a good protocol for flow cytometric analysis of surface antigens on adherent cells? And i wonder if the doublets or triplets or bigger cell clumps may interfere with positive cell population and fluorescence intensity (FITC)? Is it better to consider smaller fraction of population (~65%) composed of single cells for analysis? Thanx... gunes

Please check the website below it might help:
http://www.medicine.uiowa.edu/flowcytometry/protocols.html

You are better off looking only at the 65% or so cells, if your data requirements are stringent. There is always the risk that in excluding "clumps" (doublets, etc) you might be missing the populations of greatest interest.
On the other hand, if you are looking at cells which have undergone extended cultures, it may be useful to evaluate cell morphology, before finalizing your imaging protocols. Morphology may be highly variable in cells cultured for extended periods, even amongst "homogeneous" phenotypes.

gunes said:

Hi all, As i am new to this forum first i'd like to say "hello" to everybody... I am trying to evaluate a surface antigen (a CD one) on epithelial cells by FCM, but as i trypsinize cells to get a single cell suspension it has been realized that cells loose their surface proteins. Then i tried to detach cells by scraping, but this caused clumps. Even i passed these cells through 70 um nylon mesh, cells appeared in non-homogenous populations at FS and SS. Then i tried EDTA-PBS mixture it worked better (but not good enough to maintain a single cell suspension) but i am worrying about if EDTA interferes with antibody binding. Does anyone know a good protocol for flow cytometric analysis of surface antigens on adherent cells? And i wonder if the doublets or triplets or bigger cell clumps may interfere with positive cell population and fluorescence intensity (FITC)? Is it better to consider smaller fraction of population (~65%) composed of single cells for analysis? Thanx... gunes

There are some cell surface antigens that get destroyed by trypsininzation. Usually we just use 10mM EDTA/PBS solution to harvest those cells and sometimes you do get clumps of cells. But if you have good negative controls (negative antibody on the same cells), the FACS results will be real.