Difficulty counting PBMCs! - (May/17/2005 )
Stupid question but I am finding it difficult to count PBMCs in a trypan blue stain! There are big ones, little ones, some clumped ones... 
I know not to count the dark ones (non-viable) but can anyone give me a picture or something showing me how I can identify a mononuclear cell? (OR do I count all of the cells that are not stained??)
THANKS!
Are these purified in some way, Ficoll etc? Have you lysed the RBCs? If so, count all non-blue ones.
I have used BD vacutainer CPT tubes with sodium heparin which utilized a Ficcol Hypaque density fluid. The pamphlet states that on average there is a 28.4% RBC contamination and 5.4% Granulocyte contamination. So when I am counting the PBMCs I obviously have to differentiate between the the mononuclear cells, RBCs and granulocytes. So the question is how do I do that?
Thanks
Well you can lyse the RBCs by mixing adding acetic acid to your trypan blue solution and add that to a small sample of your cells. Use that to count your cells. RBCs will lyse and then you are left only distinguising between granulos and PBMCs. This can be done instead by subsituting the trypan blue for some other dye (gentian violet or some such) to distinguish nuclei and you can tell a PBMC from a granulo that way, like a differential count, otherwise if you have a CBC machine it will do it for you.
I have used BD vacutainer CPT tubes with sodium heparin which utilized a Ficcol Hypaque density fluid. The pamphlet states that on average there is a 28.4% RBC contamination and 5.4% Granulocyte contamination. So when I am counting the PBMCs I obviously have to differentiate between the the mononuclear cells, RBCs and granulocytes. So the question is how do I do that?
Thanks
well you are using tryphan blue, so you should lyse your cells using ammonium chloride before counting anyways.
becareful not to leave cells sitting in ammonium cholride for more than 2 minutes.
since you're dealing with monocytes, i highly suggest that after you put the 10ul of cell culture onto the hemacytometer slide, fill the tube with at least 3 mls of cDMEM+ 5% FCS and GMCSF.
you count all the cells that are not stained.
are you doing these from bone marrow preps?
becareful not to leave cells sitting in ammonium cholride for more than 2 minutes.
since you're dealing with monocytes, i highly suggest that after you put the 10ul of cell culture onto the hemacytometer slide, fill the tube with at least 3 mls of cDMEM+ 5% FCS and GMCSF.
you count all the cells that are not stained.
are you doing these from bone marrow preps?
No, I am using human whole blood and separating PBMC's from that. I basically only want PBMCs in the end with as minimal RBC's and granulocytes as possible. I will use these PBMCs for testing for immunomodulatory activity from plant extracts. I know the general method is using ACK. One method I have read about uses 30% ACK(ammonium chloride potassium) after pbmc isolation to lyse remaining red blood cells. Does anyone know if this is a good method?
THanks
[quote=MaximinaNYC,May 18 2005, 11:00 PM]
Well you can lyse the RBCs by mixing adding acetic acid to your trypan blue solution and add that to a small sample of your cells. Use that to count your cells. RBCs will lyse and then you are left only distinguising between granulos and PBMCs. This can be done instead by subsituting the trypan blue for some other dye (gentian violet or some such) to distinguish nuclei and you can tell a PBMC from a granulo that way, like a differential count, otherwise if you have a CBC machine it will do it for you.
Hi MaximaNYC. I don't have a CBC machine so manual counting is an only option! 
I basically would like to have as pure a PBMC solution after isolation (using BD CPT Vacutainer) as possible. So my main solution should also be free of RBC's and granulocytes (not just the aliquot that I place on the haematocytometer). I have a max of a 40X objective lens. I can tell what the RBC's look like (donut shaped cells) but can't tell the difference between granulo's and PBMCs. Are the granulo's the larger cells? On the microscope there are large cells (about 3-4X the size of the other 'smaller' cells) with granular cytoplasm (granulocytes??)
thanks again