A typical mouse FCM phenotypic analysis.

The following are guidelines and examples of a typical comprehensive general analysis by FCM of the major lymphoid subpopulations in mouse Bone Marrow (BM), Spleen (Spl), Thymus (Thy) and Peritoneal Cells (PerC). Other tissues such as Lymph Nodes and Peyer's Patches are not illustrated.

These should be used as guidelines. Refer to specialized papers for a more detailed analysis.
Focalize on the appearance of the cell populations (clouds of dots) in each antibody combination: you should get an identical distribution in normal mice.

BM, PerC, Thy and Spl are analyzed for Side Scatter (SSC) versus Forward Scatter (FSC). Lymphocytes, which are of low SSC and low FSC, are highlighted in red. The prominent populations to the right of the lymphocytes in BM and PerC represents myeloid cells and macrophages, respectively. Both have greater side and forward scatter properties.

click on the image to enlarge

Spleen is evaluated for B cell populations using antibodies against total B cells (B220 / CD45R), IgD, IgM, CD23, CD21.

click on the image to enlarge

As shown above, spleen cells are gated on lymphocytes either by FSC and SSC (top left), or by SSC and low FL3 fluorescence (autofluorescence)(bottom left). This latter in case you don't have any staining in this gate. If you use FL-3 for autofluorescence exclusion, make sure you compensate FL3 versus the other three fluorochromes.
You can gate with combined FSC, SSC and FL3 negative, to ensure that no autofluorescent cells score in your sample.
Lymphocytes are then gated for B220 positivity (the grey peak in the central histogram).
B220-positive B-cells are then analyzed for IgD vs IgM (top right) or CD23 vs CD21 (bottom right).

Thymus (left) and Spleen (right) are analyzed for immature and mature T lymphocyte subsets by CD4 and CD8 staining.

Peritoneal Cells are evaluated for CD5+ B cells (B1 cells). Left panel show lymphocytes gating.

click on the image to enlarge

BM is analyzed for B lymphoid precursors: staining for CD43 and B200 defines a mature B (B220+ CD43-) and an immature pro-B (B220+, CD43+; in red) populations. Similar information can be obtained by staining for A441 vs B220. This latter combination allows easy analysis of immature B cells in the spleen.

click on the image to enlarge

To download a .pdf file with this example click here or on the image.