Resting B cells were purified by forcing spleen tissue through a 40 μm mesh into complete RPMI media (Gibco) with 6% serum. Single cell suspensions were purified by magnetic cells sorting (MACS) using CD43 beads, according to the manufacturer’s instructions (Miltenyi Biotec). Indicated cell numbers were transferred intravenously into recipient mice. To assess proliferation in vitro, resting B cells purified from tTA-H2B-mCh mice were labeled at 37 °C in 5 μM carboxyfluorescein succinimidyl ester. Labeled B cells were then stimulated with LPS and IL-4 for 72 hours as previously described30 (link). DOX was added to the cultures at 500 ng/mL.
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Purification and Proliferation of Resting B Cells
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Other organizations : Rockefeller University, Howard Hughes Medical Institute
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Variable analysis
independent variables
- Doxycycline (DOX) concentration (500 ng/mL)
- B cell proliferation
- Resting B cells purified from tTA-H2B-mCh mice
- Labeling of B cells with 5 μM carboxyfluorescein succinimidyl ester
- Stimulation of B cells with LPS and IL-4 for 72 hours
- Positive control: Stimulation of B cells with LPS and IL-4
- Negative control: Not explicitly mentioned
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