cd43 bcell isolation kit  (Miltenyi Biotec)

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: Tet2 deficiency impairs memory B‐cell and plasma cell differentiation of IgG1+ germinal center B‐cells . (A) Schematic diagram of the protocol of SRBC primary and secondary immunizations. (B) Representative cytometry plot and quantification of IgM+ and IgG1+ GC B‐cells (CD19+CD95+GL7+) from Tet2 ‐WT and Tet2 ‐KO mice upon primary ( n = 8) and secondary immunization ( n = 18) with SRBC. (C) Representative cytometry plot and quantification of IgM+ and IgG1+ MBCs, (Lin (CD4, CD8, Gr1)‐IgD‐GL7‐CD19+CD38+) from Tet2 ‐WT and Tet2 ‐KO mice upon primary and secondary immunization with SRBC (primary immunization: Tet2 ‐WT [ n = 7] and Tet2 ‐KO [ n = 9]; secondary immunization: Tet2 ‐WT [ n = 9] and Tet2 ‐KO [ n = 10]). (D) Representative cytometry histograms and quantification of the expression of CD80 on the cell surface of IgM+ and IgG1+ MBCs from Tet2 ‐WT ( n = 7) and Tet2 ‐KO ( n = 9) mice upon primary immunization with SRBC. (E) Representative cytometry plot of PBs (CD19+CD138+CD43+) from Tet2 ‐WT ( n = 4) and Tet2 ‐KO ( n = 5) mice upon secondary immunization with SRBC. (F) Representative cytometry plot and quantification of IgM+ and IgG1+ PBs (CD19+CD138+CD43+) from Tet2 ‐WT ( n = 4) and Tet2 ‐KO ( n = 5) mice upon secondary immunization with SRBC. All P values were calculated using an unpaired two‐tailed t ‐test, * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, not significant, in all experiments. GC, germinal center; MBC, memory B cell; PBs, plasmablasts; SRBC, sheep red blood cells.

Article Snippet: At Day 0, splenic murine naïve B‐cells from a Tet2 ‐KO mouse were isolated from 3 to 4‐month‐old mice using the mouse B‐cell isolation kit (CD43 (Ly‐48) MicroBeads; Miltenyi Biotec) following the manufacturer's instructions (yield >90% CD19+ B220+).

Techniques: Clinical Proteomics, Cell Differentiation, Cytometry, Expressing, Two Tailed Test

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: The expansion and differentiation blockade of IgG1+ Tet2 ‐KO GC B‐cell are cell‐autonomous . (A) Schematic diagram of the in vitro differentiation system. (B) Representative cytometry plot and quantification of IgM+ and IgG1+ in vitro iGCB (CD19+ CD95+ GL7+) from Tet2 ‐WT and Tet2 ‐KO mice at D4 ( n = 18) and D8 ( n = 15). (C) Schematic illustration of the in vitro culture system of iGCB expressing a specific Ig isotype. (D) Number of live Tet2 ‐WT and Tet2 ‐KO B‐cells cultured on 40LB with IL21 on Day 8 (D8) ( n = 4). (E) Representative cytometry plots of iPC on Day 8. (F) Quantification of flow cytometry data corresponding to D8 iPC ( n = 4). (G) Number of live iPC (CD19+ CD138+) Tet2 ‐WT and Tet2 ‐KO B‐cells on Day 8 (D8) ( n = 4). All P values were calculated using an unpaired two‐tailed t ‐test, * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, not significant, in all experiments. D, Day; iGCB, induced germinal center B‐cells, iPC, induced plasma cells; SRBC, sheep red blood cells.

Article Snippet: At Day 0, splenic murine naïve B‐cells from a Tet2 ‐KO mouse were isolated from 3 to 4‐month‐old mice using the mouse B‐cell isolation kit (CD43 (Ly‐48) MicroBeads; Miltenyi Biotec) following the manufacturer's instructions (yield >90% CD19+ B220+).

Techniques: In Vitro, Cytometry, Expressing, Cell Culture, Flow Cytometry, Two Tailed Test, Clinical Proteomics

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: Tet2 deficiency induces higher c‐Rel activity in IgG1+ germinal center B‐cells . (A , B) Representative cytometry plots (A) and quantification (B) of intracellular and nuclear localization of c‐Rel and phosphorylated p65 (Ser536) in sorted IgM+ and IgG1+ Tet2 ‐WT and Tet2 ‐KO B cells at D4 of the in vitro GC model ( n = 3). (C , D) Representative cytometry plots (C) and quantification (D) of intracellular and nuclear localization of c‐Rel and phosphorylated p65 (Ser536) in sorted IgG1+ Tet2 ‐WT and Tet2 ‐KO B cells at D8 of the in vitro GC model ( n = 3). (E) Expansion factor compared to empty MSCV condition between D11 and D14 and quantification of MFI of IgG1 in IgG1+ iGCB (GFP+ CD19+ CD138− Fas+ GL7+ IgG1+) at D14 of the iGCB transduction model, after transduction of Tet2 ‐KO B‐cells by an empty MSCV, a MSCV containing shRNA Rel 908, or a MSCV containing shRNA Rel 1869 ( n = 3). For (B , D) , P values were calculated using an unpaired two‐tailed t ‐test. For (E) , P values were calculated using a paired RM one‐way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, not significant in all experiments. ANOVA, analysis of variance; GC, germinal center; iGCB, induced germinal center B‐cell; MFI, mean fluorescence intensity; MSCV, murine stem cell virus.

Article Snippet: At Day 0, splenic murine naïve B‐cells from a Tet2 ‐KO mouse were isolated from 3 to 4‐month‐old mice using the mouse B‐cell isolation kit (CD43 (Ly‐48) MicroBeads; Miltenyi Biotec) following the manufacturer's instructions (yield >90% CD19+ B220+).

Techniques: Activity Assay, Cytometry, In Vitro, Transduction, shRNA, Two Tailed Test, Fluorescence, Virus