mouse spleen cells

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Beru
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mouse spleen cells

I am optimizing a protocol to sort T cells from mice spleen cells using CD90 microbeads and analyze CD3+ cells using flow cytometry. The total population of T cells in the spleen was 41%(A); and when I do magnetic sorting using CD90 microbeads I got 91% CD3+ T cells (B). Then when I got a new group of mice, the T cell population goes down to 21-25%(C) and magnetic separation gave me only 53% (D) and later 71-75% (E) CD3+ cells. When checking lung swab, actually we isolated Staphylococcus and Bacillus from the old group of mice where the T cells >41% (A). That explains why I got fewer T cell (around 23%) (C) in the new group ("healthy group") but that doesn't help me explain why I got fewer CD3+ cells (also more - 10% B220+ cells) in CD90 microbead isolated cells (D & E). I also have some problem with non stained and stained cells.. (F & G) - linear lined cells in both FL1 and FL2 Vs FL1. I gated those cells and they are all over (H). Thank you for your help.
Beru