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. Author manuscript; available in PMC: 2010 Nov 20.
Published in final edited form as: Immunity. 2009 Nov 5;31(5):749–760. doi: 10.1016/j.immuni.2009.08.026

Figure 4. MD4ML5 PTEN-deficient B cells exhibit low receptor occupancy.

Figure 4

A) Surface HEL staining of MD4 (green), MD4ML5 (blue) and MD4ML5PTENflox/flox (red) B cells was determined by flow cytometry. B) (Upper, left panels), MD4, MD4ML5, and MD4ML5PTENflox/flox splenic cells from adult mice (> 8 weeks old) and (lower, left panels) young mice (3 weeks old) were incubated in 20 μg/ml HEL (blue) or PBS (red). Surface HEL was detected using polyclonal antibody against HEL and measured by flow cytometry. Right panel, Percent receptor occupancy was determined from the ratio of HEL staining intensity of PBS-incubated cells to HEL-incubated cells. The asterisk (*) indicates a p value of < 0.05. C) MD4 B cells were CFSE-labelled and equal cell numbers were transferred into MD4ML5 (blue), MD4ML5PTENflox/flox (red), MD4PTENflox/flox (dotted) and MD4 (black) recipient mice by tail vein injection. After 24 hr, recipient splenic cells were harvested, stained with anti-B220 and anti-IgM. CFSE+ donor B cells were gated, and surface IgM was analyzed by flow cytometry. D). MD4, MD4PTENflox/flox and MD4ML5PTENflox/flox mice were injected with 1 mg of HEL (blue), 3 mice per group or PBS (red), one mouse per group. Splenic cells were isolated at 48 hr post-injection, enumerated and examined for expression of B220 by flow cytometry. Histograms represent B220 gated cells.