I'm having difficulty to choose the appropriate cell population in the fsc area vs ssc area plot for further gating and analysis. I have prepared the sample (single cell suspension) from mouse spleen which was stored in minus 86 degree celcius fridge. Standard RBC lysis and washing proceedure was followed to prepare and stain cells with two different panel of antibodies. Unstained cells and excess antibidies were washed out using 500 microlitre of facs buffer. Then stained cells were resuspended in sheath fluid(miliqeu water) and subjected to flow cytometry ( Beckman Coulter) The cell population I'm having is so scattered in throughput the plot. As we know due to less complexity lymphocytes tend to scatter forward light most, they should appear in the FSC region in cluster. Sadly I'm not getting such cluster. Dear researchers I seek some suggestion from all of you in this regard. Thank you in advance.
Nazmul Hasan
Dear Nazmul Hasan,
I suppose the first step you should take is to observe the cells with a microscope.
Because the base mechanism of flow cytometry is identical to microscopy (you can see FSC), the microscopic observation should be relevant to flow cytometric data. If they are consistent, this could mean that cells are in a bad condition already after thawing. If you find any inconsistency between the miscroscopic and flow cytometric observations, this could mean a problem in your methods for flow cytometric analysis.
Dear Tetsuo Tsukamoto
Thank you very much for your valuable suggestion. I will follow your guidelines accordingly.
As Tetsuo suggested, observe the cells under a microscope.
Check the dilution and pH of RBC lysis buffer. Incorrect dilution or pH can affect the lymphocytes. It may not be a good idea to resuspend cells in sheath fluid. We always resuspend ours in FACS buffer (PBS with 1 mM EDTA and 1% FBS. If you are using frozen cells, it helps having DNAse in the thawing buffer.
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