I separated the mononuclear cells from the spleen of the mouse and I want to measure different strains of the lymphocytes by using flow cytometric technique, but the number of cells is very low.
Hi,
I don't know any strategy to increase your cell recovery rates, if that is the case. However, if the problem is that you have low numbers of lymphocytes in a sample (say 1-3% lymphocytes), it should not represent a problem for FACS evaluations as long as you acquire more than 200, 000 events. You would be able to analyze the phenotype of those few lymphocytes. I have no experience with spleen, but I have worked with human samples enriched of macrophages (less than 5% lymphocytes) and they are analizable. If you need to analyze many markers or lymphocyte subpopulation then you would need to acquire over 300,000 events.
Good luck.
What protocol are you following for cell isolation in the spleen? Our isolation protocol usually yields upwards of 20 million cells, which should be more than enough for FC analysis. Mitogens like CONA and PHA are relatively specific for T cells, so they'll likely cause expansion of that specific population over others -- B cells and NK cells, for example. CONA will actually likely cause agglutination of many of the cell types in the spleen, and may actually render samples more difficult to perform flow cytometry on.
If you tell us a little more about your experimental design and end goals, perhaps we can make more useful suggestions.
You might be analyzing the cell population which accounts to small numbers.
I would recommend to gate the population you are interested and acquire more than 2,000 event of that population.
Spleen of WT mice usually yields around 40-100 millions cells after RBC lysis. Using PHA or other mitogen will activate your cells and would be important if you are looking for cytokines. PHA stimulation for 4 hrs leads to significant cell death also.
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