I stained the cells with mouse CD45.2-PE antibody to the single cell suspension prepared from mouse bone marrow. Even after adding twice the recommended concentration, I am still seeing the unstained population in my FACS data. My reaction volume is 100ul, with 10E6 cells and 1ug antibody. Any idea..?
Few points to consider.
Firstly which mouse strain are you working with? There are CD45.1 and CD45.2 specific strains like nicely illustrated in the previous comment.
Second, what is the % of your unstained cells in the blood compared to the bone marrow? You can have some CD45 negative cells in the bone marrow i.e stromal cells, mesenchymal stem cell etc. The real problem is if you have a discrete % of this unstained population also in the peripheral blood.
In any case, if you don`t have to specifically use a CD45.2 I would get a pan CD45 Ab as first troubleshooting.
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