Isolation of B Cell Subsets from Bone Marrow

Single-cell suspensions of B220+ B cells from BM were incubated with fluorescently labeled antibodies specific for CD43, B220, IgM, and CD23 in a staining buffer (PBS with 0.5% BSA) for 20 min at 4 °C. Further, the cells were incubated with DAPI to select living cells (DAPI−) and washed, then pro-B (B220+CD23-CD43+IgM–), pre-B (B220+CD23–CD43–IgM–), and immature B cells (B220+CD23–CD43–IgM+) were isolated [11 (link)]. Cell sorting was performed using a FACS Influx Sorter (BD Biosciences). The purity of sorted cells ranged from 95% to 98%.

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Flores-Fernández R., Aponte-López A., Suárez-Arriaga M.C., Gorocica-Rosete P., Pizaña-Venegas A., Chávez-Sanchéz L., Blanco-Favela F., Fuentes-Pananá E.M, & Chávez-Rueda A.K. (2021). Prolactin Rescues Immature B Cells from Apoptosis-Induced BCR-Aggregation through STAT3, Bcl2a1a, Bcl2l2, and Birc5 in Lupus-Prone MRL/lpr Mice. Cells, 10(2), 316.

Publication 2021

FACS Influx Sorter

Antibodies B cells Buffer Cd43 Dapi Immature b cells

Corresponding Organization : Hospital Infantil de México Federico Gómez

Other organizations : Universidad Nacional Autónoma de México, Instituto Politécnico Nacional, Instituto Nacional de Enfermedades Respiratorias

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Variable analysis

independent variables

Incubation of B220+ B cells from BM with fluorescently labeled antibodies specific for CD43, B220, IgM, and CD23

dependent variables

Identification and isolation of pro-B (B220+CD23-CD43+IgM–), pre-B (B220+CD23–CD43–IgM–), and immature B cells (B220+CD23–CD43–IgM+)

control variables

Staining buffer (PBS with 0.5% BSA)
Incubation time (20 min)
Incubation temperature (4 °C)
Use of DAPI to select living cells (DAPI−)

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