B Cell Isolation and Characterization

BM was collected from WT, Igκ^del, κ-mac, κ-mac Cxcr4^fl/flmb1-cre− or κ-mac Cxcr4^fl/flmb1-cre+ mice and cells were resuspended in staining buffer: (3% (v/v) fetal bovine serum (FBS) in 1x phosphate buffered saline. Erythrocytes were lysed with ACK lysis buffer (Lonza cat # 10-548E) and cells were stained with rat anti-CXCR4 (2B11), rat anti-CD43 (S7), rat anti-IgM (R6–60.2), rat anti-IgD (11 (link)-36 (link)), rat anti-CD19 (1D3) and rat anti-B220 (RA3-6B2l), (all from BD Biosciences) as described previously (77 (link), 78 (link)) and viability dye eFluor 506 (eBioscience). Pre-pro-B cells (CD19−B220+IgM−), pro-B cells (CD19+B220+CD43+IgM−), large pre-B cells (B220+CD43−IgM−FSChi), small pre-B cells (B220+CD43− IgM−FSC^low) and immature B cells (B220+CD43−IgM+) were isolated by cell sorting with a FACSAria II (BD Biosciences).

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Okoreeh M.K., Kennedy D.E., Emmanuel A.O., Veselits M., Moshin A., Ladd R.H., Erickson S., McLean K.C., Madrigal B., Nemazee D., Maienschein-Cline M., Mandal M, & Clark M.R. (2022). Asymmetrical forward and reverse developmental trajectories determine molecular programs of B cell antigen receptor editing. Science immunology, 7(74), eabm1664.

Publication 2022

ACK lysis buffer eFluor 506 FACSAria II

Anti igd Anti igm Buffer Cd43 Cells Cxcr4 Erythrocytes Fetal bovine serum Immature b cells Mice Phosphate Pre b cells Pro b cells Saline

Corresponding Organization : University of Chicago

Other organizations : Scripps Research Institute, University of Illinois at Chicago

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Variable analysis

independent variables

Genotype: WT, Igκ^del, κ-mac, κ-mac Cxcr4^(fl/fl) mb1-cre− or κ-mac Cxcr4^(fl/fl) mb1-cre+

dependent variables

Percentages of pre-pro-B cells (CD19−B220+IgM−), pro-B cells (CD19+B220+CD43+IgM−), large pre-B cells (B220+CD43−IgM−FSC^hi), small pre-B cells (B220+CD43− IgM−FSC^low) and immature B cells (B220+CD43−IgM+)

control variables

Cell staining with rat anti-CXCR4 (2B11), rat anti-CD43 (S7), rat anti-IgM (R6–60.2), rat anti-IgD (11 (link)-36 (link)), rat anti-CD19 (1D3) and rat anti-B220 (RA3-6B2l) antibodies
Cell viability assessed with eFluor 506 (eBioscience)
Cell sorting with a FACSAria II (BD Biosciences)

controls

No positive or negative controls were explicitly mentioned.

Annotations

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