Journal: Cell reports

Article Title: Loop extrusion promotes an alternate pathway for isotype switching

doi: 10.1016/j.celrep.2021.110059

Figure Lengend Snippet: B cells from M1′GFP + (A–D) or C57BL/6 (E–G) mice were activated with NOT or LPS, respectively. B cell cultures were from at least three independent mice and all PCR assays were tested in triplicate. (A and E) (Left panels) Representative plots of B cells were gated for IgM + and IgM − and then for IgG1 and IgE (A), or IgG3 and IgG2b (E) by flow cytometry on day 3 of cell stimulation. (Right panels) The frequency of IgG1 + and IgE + (A) or IgG3 + and IgG2b + (E) B cells in IgM + (red bar) and IgM − (blue bar) subpopulations was quantitated; SEMs are shown. Mice tested (n). P values (0.001, ***) from two tailed Student’s t test. (B–D) B cells activated with NOT for 4 days were purified using anti-IgM beads, and IgM + (bound) and IgM − (flowthrough) B cells were collected. (B) Cells were FACS purified based on surface IgM, IgG1, and GFP; the percentages of cells in each quadrant are noted. (C) qRT-PCR for PSTs, dPSTs, and AID. (D) B cells were analyzed by flow cytometry for surface IgM and IgG1 and cytoplasmic for IgE. The percentage of IgE + B cells is a fraction of each cell subset. (F) IgM − B cells (flowthrough) were further purified by FACS based on surface IgM, IgG3, and IgG2b. The percentage of IgM − , IgM − IgG3 + , and IgM − IgG2b + cells is shown. (G) qRT-PCR was performed on cDNAs prepared in (F).

Article Snippet: B cells were activated with NOT or LPS for 40 hours, 3- and 4 days and then IgM + (bound) and IgM dim /IgM − (flowthrough) B cells were isolated using anti-IgM magnetic microbeads (MACS, Miltenyi) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Cell Stimulation, Two Tailed Test, Purification, Quantitative RT-PCR

Journal: Cell reports

Article Title: Loop extrusion promotes an alternate pathway for isotype switching

doi: 10.1016/j.celrep.2021.110059

Figure Lengend Snippet: All B cell cultures were derived from at least three independent mice. (A) C57BL/6 resting splenic B cells were activated with LPS for 3 days, and IgM + (bound) and IgM dim /IgM − (flowthrough) B cells were isolated and analyzed using anti-IgM-eFluor 450 (e450), anti-IgG3-Brilliant Violet 510 (BV510), and anti-IgG2b-FITC. (Left panels) IgM + IgG3 − IgG2b − cells (i), IgM dim IgG3 − IgG2b − cells (ii), andIgM − IgG3 − IgG2b − cells (iii) were FACS purified and re-cultured in LPS for 24 h (orange arrows). (Middle panels) Re-cultured cells were analyzed for IgM + , IgM − , and then for IgG3 + , IgG2b + cells. (Right panels) Quantitation of IgG3 + and IgG2b + B cells from IgM + and IgM − subsets with SEMs shown. (B) FACS gating strategy to isolate IgM − IgG3 − IgG2b − IgD − Igκ − (BCR neg ) and IgM − IgG3 − IgG2b − IgD + Igκ + (IgD + Igκ + ) B cells. BCR neg (orange arrow) and IgD + Igκ + (blue arrow) were re-cultured in LPS for 24 h and analyzed for surface IgM, IgG3, and IgG2b. (C) B cells dynamically transition between IgM + and BCR neg states.

Article Snippet: B cells were activated with NOT or LPS for 40 hours, 3- and 4 days and then IgM + (bound) and IgM dim /IgM − (flowthrough) B cells were isolated using anti-IgM magnetic microbeads (MACS, Miltenyi) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Isolation, Purification, Cell Culture, Quantitation Assay

Journal: Cell reports

Article Title: Loop extrusion promotes an alternate pathway for isotype switching

doi: 10.1016/j.celrep.2021.110059

Figure Lengend Snippet: (A–C) Results are representative of least three independent experiments. C57BL/6 (A–E) AID −/− and 53BP1 −/− (E) resting splenic B cells were activated with LPS for 3 days and IgM dim /IgM − (flowthrough) B cells were isolated (A–D). (A) FACS gating strategy to purify IgM dim IgD − CD138 − and BCR neg CD138 − cells using anti-IgM (e450), anti-IgD (phycoerythrin [PE]), anti-IgG3/anti-IgG2b (FITC), anti-Igk (PE-CF594), and anti-CD138 (PE-Cy7). (B) IgM dim IgD − CD138 − and BCR neg CD138 − cells were stained with Vybrant DyeCycle violet stain and cell cycle phases are shown. (C) IgM dim IgD − CD138 − and BCR neg CD138 − B cells were fixed, permeabilized, stained with anti-B220 (PE, red), anti-IgM (e450, blue), and anti-IgG3/anti-IgG2b (FITC, green), and examined by fluorescence microscopy. Scale bars, 10 μm. (D) qRT-PCR for transcripts from BCR neg CD138 − and IgM dim IgD − CD138 − B cells and PCR assays were tested in triplicate and averaged, and SEMs are shown. (E) Activated B cells were gated for CD138 − IgM − IgD − IgG3IgG2b − Igκ − Igλ − (CD138 − BCR neg ) B cells. (F) The frequency of CD138 − BCR neg B cells is calculated as a percentage and was an average of live cells from three independent experiments with SEMs shown. P ≤ values (p % 0.05, *) from two tailed Student’s t test.

Article Snippet: B cells were activated with NOT or LPS for 40 hours, 3- and 4 days and then IgM + (bound) and IgM dim /IgM − (flowthrough) B cells were isolated using anti-IgM magnetic microbeads (MACS, Miltenyi) according to the manufacturer’s instructions.

Techniques: Isolation, Staining, Fluorescence, Microscopy, Quantitative RT-PCR, Two Tailed Test

Journal: Cell reports

Article Title: Loop extrusion promotes an alternate pathway for isotype switching

doi: 10.1016/j.celrep.2021.110059

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: B cells were activated with NOT or LPS for 40 hours, 3- and 4 days and then IgM + (bound) and IgM dim /IgM − (flowthrough) B cells were isolated using anti-IgM magnetic microbeads (MACS, Miltenyi) according to the manufacturer’s instructions.

Techniques: Recombinant, Staining, Knock-In, Software