cd43 b cells  (Miltenyi Biotec)

Journal: Toxicology Reports

Article Title: Genotoxicity risk assessment of a 7-hydroxymitragynine-enriched Kratom preparation: An integrated in silico and in vitro approach

doi: 10.1016/j.toxrep.2026.102206

Figure Lengend Snippet: Micronucleus test of Kratom leaf extract after 4 h exposure with S9 in TK6 cells. Results are the mean ± SD of 3 independent experiments. Statistical testing with one-way ANOVA and Tukey’s post-hoc test (* p < 0.05).

Article Snippet: The human B lymphoblastoid cell line (TK6) (CRL-8015; batch No. 70045146), purchased from ATCC, was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin.

Techniques:

Journal: Toxicology Reports

Article Title: Genotoxicity risk assessment of a 7-hydroxymitragynine-enriched Kratom preparation: An integrated in silico and in vitro approach

doi: 10.1016/j.toxrep.2026.102206

Figure Lengend Snippet: Micronucleus test of Kratom leaf extract after 4 h exposure without S9 in TK6 cells. Results are the mean ± SD of 3 independent experiments. Statistical testing with one-way ANOVA and Tukey’s post-hoc test (* p < 0.05).

Article Snippet: The human B lymphoblastoid cell line (TK6) (CRL-8015; batch No. 70045146), purchased from ATCC, was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin.

Techniques:

Journal: Toxicology Reports

Article Title: Genotoxicity risk assessment of a 7-hydroxymitragynine-enriched Kratom preparation: An integrated in silico and in vitro approach

doi: 10.1016/j.toxrep.2026.102206

Figure Lengend Snippet: Micronucleus test of Kratom leaf extract after 24 h exposure without S9 in TK6 cells. Results are the mean ± SD of 3 independent experiments. Statistical testing with one-way ANOVA and Tukey’s post-hoc test (* p < 0.05).

Article Snippet: The human B lymphoblastoid cell line (TK6) (CRL-8015; batch No. 70045146), purchased from ATCC, was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: In vitro culture conditions maintaining the naïve B cell state. (A) Schematic diagram of the different steps of the in vivo CRISPR/Cas9 screening system. Briefly, naïve B cells isolated from donor mice of the indicated genotype are transduced with LV particles expressing sgRNAs specific for the genes of interest and a neutral control gene. After infection, the cells are cultured in vitro for 3 days. Transduced mCherry + donor B cells are sorted by flow cytometry and transferred to recipient mice of the indicated genotype, which are immunized with the hapten NP to induce an immune response. The resulting mCherry + plasmablasts are isolated, and the abundance of sgRNAs in plasmablasts is determined and compared with the abundance of the sgRNAs in the donor B cells. (B) Flow cytometric analysis of wild-type naïve splenic B cells cultured in vitro for 3 days under different conditions. B cells were cultured either in the B cell medium alone, or additionally in the presence of LPS, sBAFF, OP9 cells, or OP9 cells plus sBAFF. Before starting the culture, B cells were labeled with the CellTrace Violet dye. After 3 days, the cell viability, CD69 and IgD cell surface protein expression, differentiation into plasmablasts (CD138 + CD22 – ), and cell proliferation were assessed. (C) Summary of all the data generated with the different culture conditions indicated. The statistical data are shown as mean values with SEM and were analyzed by one-way ANOVA with Tukey’s multiple comparisons test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Each dot corresponds to one mouse. Independent experiments were performed from two (IgD) to 10 (viability) times.

Article Snippet: CD43 – B cells were enriched from the spleen or lymph nodes of mice by immunomagnetic depletion of non-B cells using CD43 (Ly-48) MicroBeads (Miltenyi Biotec).

Techniques: In Vitro, In Vivo, CRISPR, Isolation, Transduction, Expressing, Control, Infection, Cell Culture, Flow Cytometry, Labeling, Generated

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: Characterization of the Rosa26 LSL-EcoR allele. (A) Schematic diagram and expression of the Rosa26 LSL-EcoR allele. SLC7A1 (EcoR) protein expression was analyzed by flow cytometry in mature B cells from Cd23 -Cre Rosa26 LSL-EcoR/+ and control Cd23 -Cre Rosa26 +/+ mice and is displayed as a histogram or quantification of the geometric mean fluorescence intensity (gMFI). Statistical data are shown as mean values with SEM and were analyzed by the unpaired t test; ***P < 0.001. Each dot represents one mouse. (B) Flow cytometric analysis of B cells from Cd23 -Cre Rosa26 LSL-EcoR/+ and control Cd23 -Cre Rosa26 +/+ mice, which were transduced with ecotropic LV particles expressing the mCherry fluorescent reporter protein (mCherry-LV) and were subsequently cultured in vitro for 3 days in the presence of OP9 cells with sBAFF. The percentage of transduced mCherry + B cells is shown. The bar graph indicates the transduction efficiency relative to all B cells. Statistical data are shown as mean values with SEM and were analyzed by the multiple unpaired t test with Holm–Šídák’s correction; ****P < 0.0001. Two experiments were performed, and each dot corresponds to one mouse. (C) Flow cytometric analysis of splenic B cells from Cd23 -Cre Rosa26 LSL-EcoR/+ and control Cd23 -Cre Rosa26 +/+ mice. The frequencies are shown for total B cells (CD19 + B220 + ), immature B cells (CD19 + CD93 + ), T1 B cells (CD19 + CD93 + IgM hi CD23 − ), T2 B cells (CD19 + CD93 + IgM hi CD23 + ), mature B cells (CD19 + CD93 − ), MZ B cells (CD19 + CD93 − CD21 hi Cd23 −/lo ), FO B cells (CD19 + CD93 − CD21 lo CD23 hi ), GC B cells (CD19 + B220 + GL7 + CD95 + ), and plasma cells (CD138 + TACI + ). (D) Flow cytometric analysis of bone marrow plasma cells from Cd23 -Cre Rosa26 LSL-EcoR/+ and control Cd23 -Cre Rosa26 +/+ mice. (E) Quantification of the number of total B, immature B, mature B, GC B, and plasma cells in the spleen, and plasma cells in the bone marrow from Cd23 -Cre Rosa26 LSL-EcoR/+ and control ( Cd23 -Cre Rosa26 +/+ and Rosa26 +/+ ) mice. Statistical data are shown as mean values with SEM. All pairwise comparisons are nonsignificant, as analyzed by the multiple unpaired t test with Holm–Šídák’s correction test. Two (A and B) and three (C–E) independent experiments were performed. Each dot represents one mouse.

Article Snippet: CD43 – B cells were enriched from the spleen or lymph nodes of mice by immunomagnetic depletion of non-B cells using CD43 (Ly-48) MicroBeads (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Control, Fluorescence, Transduction, Cell Culture, In Vitro, Clinical Proteomics

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: Description and characterization of different aspects of the sgRNA screening system. (A) Flow cytometric analysis of mCherry expression in B cells at 2 and 3 days after infection with a control mCherry-LV. The cells were cultured on OP9 cells with sBAFF (right). The frequencies of mCherry + B cells are shown as mean values with SEM and were analyzed by one-way ANOVA with Tukey’s multiple comparisons test; ***P < 0.001; ****P < 0.0001. (B) Flow cytometric analysis of dividing and nondividing mCherry + B cells after 3 days in culture on OP9 cells with sBAFF. Mature CD43 – B cells were incubated with CellTrace Violet prior to infection with a control mCherry-LV and were subsequently cultured for 3 days. The frequencies of mCherry + B cells among the dividing and nondividing B cell population are shown as mean values with SEM and were analyzed by the unpaired t test; ***P < 0.001. (C) Immunofluorescence staining of spleen sections from recipient mice at 5 and 7 days after immunization with NP-KLH/alum. LV infection and B cell transfer were performed as described in D. Spleen sections were stained with antibodies against IgD (green), mCherry (red), TCRβ (blue), and IRF4 (white). A nonimmunized mouse, which did not receive transduced mCherry + B cells, was used as a control. The scale bar represents 100 μm. (D) Time course of plasmablast formation upon immunization with NP-Ficoll. Naïve B cells isolated from the donor mice were transduced with LV particles expressing a neutral control sgRNA (sg. Chr1 ) and the mCherry reporter protein. After infection, B cells were cultured in vitro for 3 days in the presence of stromal OP9 cells and sBAFF. Transduced mCherry + B cells were isolated and transferred to recipient mice, which were immunized 16 h later with NP-Ficoll in PBS. Flow cytometric analysis of splenocytes from recipient mice at day 3, 5, and 7 after immunization (left) revealed the percentages of mCherry + B cells in total splenocytes and mCherry + plasmablasts (TACI + CD138 + ) within the mCherry + cells, as shown in the bar graphs (right). Absolute numbers of the mCherry + B cells and mCherry + plasmablasts in the spleen are also indicated (far right). Statistical data are shown as mean values with SEM and were analyzed by the Welch ANOVA with the Brown–Forsythe test; *P < 0.05; **P < 0.01; ****P < 0.0001. Each dot represents one mouse. AF, autofluorescence measured in the BV605 channel. (E) Scheme describing the steps taken for the selection of the 379 genes to be studied in the in vivo CRISPR/Cas9 screening experiments. The data shown in A, B, and D are based on two independent experiments. Each dot corresponds to one mouse.

Article Snippet: CD43 – B cells were enriched from the spleen or lymph nodes of mice by immunomagnetic depletion of non-B cells using CD43 (Ly-48) MicroBeads (Miltenyi Biotec).

Techniques: Expressing, Infection, Control, Cell Culture, Incubation, Immunofluorescence, Staining, Isolation, Transduction, In Vitro, Selection, In Vivo, CRISPR

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: Testing of the in vivo model system used for CRISPR/Cas9 screening experiments. (A) Schematic diagram of the setup used for testing the in vivo CRISPR/Cas9 screening system. Naïve B cells isolated from donor mice were transduced with LV particles expressing a neutral control sgRNA and the mCherry reporter protein. After infection, B cells were cultured in vitro for 3 days in the presence of OP9 cells and sBAFF. Transduced mCherry + B cells were sorted by flow cytometry and transferred to recipient mice, which were immunized 16 h later with NP-KLH in alum. mCherry + plasma cells and mCherry + GC B cells were analyzed by flow cytometry at different time points after immunization. (B) Naïve B cells of the donor genotype were infected with a mCherry-LV expressing a neutral control sgRNA and sorted followed by transfer to mice of the recipient genotype and NP-KLH immunization, as described in A. Flow cytometric analysis of splenocytes from recipient mice was performed 3, 5, 7, and 10 days after immunization. The percentages of total mCherry + B cells in total splenocytes, mCherry + plasmablasts (TACI + CD138 + ), and mCherry + GC B cells (GL7 + CD95 + ) within the mCherry + B cell population (left), and the absolute numbers of these cells (right) are indicated. AF, autofluorescence measured in the BV605 channel. (C) Naïve B cells isolated from donor mice were transduced with LV particles expressing a control sgRNA (sg. Chr1 ) or sgRNAs targeting Prdm1 or Irf4 . After infection, B cells were cultured in vitro for 3 days and transduced mCherry + B cells were transferred to recipient mice followed by immunization with NP-KLH, as described in A. 6 days after immunization, the splenocytes were analyzed by flow cytometry. The percentages of total mCherry + B cells, mCherry + plasmablasts, and mCherry + GC B cells among total B cells are indicated. Statistical data (B and C) are shown as mean values with SEM and were analyzed by the multiple unpaired t test with Holm–Šídák’s correction test; *P < 0.05; **P < 0.01; ****P < 0.0001. Two independent experiments (B and C) were performed. Each dot represents one mouse.

Article Snippet: CD43 – B cells were enriched from the spleen or lymph nodes of mice by immunomagnetic depletion of non-B cells using CD43 (Ly-48) MicroBeads (Miltenyi Biotec).

Techniques: In Vivo, CRISPR, Isolation, Transduction, Expressing, Control, Infection, Cell Culture, In Vitro, Flow Cytometry, Clinical Proteomics

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: Indel sequencing data, identification of GC B cell– and PB-specific sgRNA hits, and sgRNA representation at different stages of the screening experiments. (A and B) Indel sequencing. Mature CD43 – B cells were infected with the indicated sgRNA mCherry-LVs, cultured for 3 days on OP9 cells with sBAFF, and then stimulated with CpG, IL-4, and IL-5 for another 3 days, followed by flow cytometric sorting of the mCherry + B cells and DNA preparation. Indel sequencing was performed by PCR amplification and sequencing of a DNA fragment spanning the sgRNA break site, followed by TIDE analysis . (A) Percentages of indels are indicated relative to the break site of sg. Cd44 (position 0, black), demonstrating that the percentage of indels is 88% for the sg. Cd44 . (B) Percentages of indels are shown for the indicated sgRNAs, as determined by the indel sequencing and TIDE analysis. (C) Identification of GC B cell–specific and PB-specific positive and negative regulators, as determined by CRISPR/Cas9 screening experiments at day 7 after NP-KLH immunization. The log 2 FC plot (left) indicates the sgRNA hits that were determined by a more than twofold change in sgRNA abundance in GC B cells and PBs (corresponding to ). The genes corresponding to the GC B cell–specific and PB-specific sgRNA hits are shown (right), and their fold changes and P values are indicated in . The common positive regulators are shown in , as being significant in both cell types, while the PB-specific positive regulators are indicated as being nonsignificant in the GC B cell analysis . (D) sgRNA representation at different stages of the TD and TI screening experiments. Flow cytometric analysis was used to determine the number of sorted mCherry + B cells at the start, the splenic mCherry + B cells at day 3 after B cell transfer ( [TD] and S1D [TI]), and the splenic mCherry + PBs, GC B cells, and memory B cells at day 7 ( [TD] and S4D [TI]). As the screening library contained 882 sgRNAs, the number of identified B cells was divided by 882 to determine how many cells contained one specific sgRNA (cells/sgRNA) under the assumption that each cell was only infected by one sgRNA virus (MOI = 1). (E) Schematic representation of the LV vectors used in this study. LVs containing the PGK-mCherry or EF1a-mCherry gene were used for establishing and testing of the in vivo CRISPR/Cas9 screening system. The sgRNA library was cloned in a LV vector containing the EF1as-mCherry gene. The validation experiments were performed with LVs containing the EF1as-mCherry or EF1as-mAmetrine gene. 5′LTR, 5′ long terminal repeat; Ψ, psi packaging signal; RRE, Rev response element; cPPT, central polypurine tract; PBS, primer binding site for DNA sequencing library preparation; hU6, human U6 promoter; hPGK, human phosphoglycerate kinase promoter; hEF1a, human elongation factor 1a promoter; hEF1as, human elongation factor 1a short promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; 3′LTR (SIN), 3′ long terminal repeat (self-inactivating); FC, fold change; PBs, plasmablasts.

Article Snippet: CD43 – B cells were enriched from the spleen or lymph nodes of mice by immunomagnetic depletion of non-B cells using CD43 (Ly-48) MicroBeads (Miltenyi Biotec).

Techniques: Sequencing, Infection, Cell Culture, Amplification, CRISPR, Cell Analysis, Virus, In Vivo, Clone Assay, Plasmid Preparation, Biomarker Discovery, Binding Assay, DNA Sequencing

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: CRISPR/Cas9 screen for identifying new regulators of the TD B cell responses. (A) Schematic diagram of the in vivo CRISPR/Cas9 screening experiments used to study the TD B cell responses. Naïve B cells isolated from donor mice were transduced with LV particles, each expressing one of the 882 sgRNAs and the mCherry reporter protein. After infection, B cells were cultured in vitro for 3 days in the presence of OP9 cells and sBAFF. Transduced mCherry + donor B cells were sorted by flow cytometry and transferred to recipient mice, which were immunized 16 h later with NP-KLH/alum. 7 days after immunization, splenic mCherry + CD45.2 + cells were enriched by anti-CD45.1 antibody–mediated MACS depletion of the CD45.1 + CD45.2 + recipient cells. Plasmablasts and GC B cells were isolated from the enriched fraction by flow cytometric sorting, followed by DNA extraction, library preparation, and DNA sequencing. DNA was also extracted from a fraction of the sorted transduced mCherry + donor B cells before immunization, followed by library preparation and DNA sequencing. (B) Flow cytometric analysis of the enriched mCherry + CD45.2 + B cells from recipient mice. The gates used for the sorting of plasmablasts (TACI + CD138 + ) and GC B cells (CD19 + GL7 + CD95 + ) are indicated. The percentages of mCherry + plasmablasts and mCherry + GC B cells within the sorted cell population are shown. (C) Numbers of sorted plasmablasts and GC B cells (left), the percentage of mCherry + plasmablasts and mCherry + GC B cells within the sorted cell population (middle), and the numbers of mCherry + plasmablasts and mCherry + GC B cells (right) were determined for individual recipient mice analyzed in two independent screening experiments. (D) Total sgRNA representation in each plasmablast and GC B cell replicate sample analyzed in two screening experiments. The sgRNA representation was estimated by dividing the number of mCherry + plasmablasts or GC B cells by the number of the 882 sgRNAs constituting the sgRNA library. Each plasmablast and GC B cell replicate sample contained cells that were obtained from five or six recipient mice after pooling. Each colored box represents the individual contribution of each mouse to the sgRNA representation of the entire replicate sample.

Article Snippet: CD43 – B cells were enriched from the spleen or lymph nodes of mice by immunomagnetic depletion of non-B cells using CD43 (Ly-48) MicroBeads (Miltenyi Biotec).

Techniques: CRISPR, In Vivo, Isolation, Transduction, Expressing, Infection, Cell Culture, In Vitro, Flow Cytometry, DNA Extraction, DNA Sequencing

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: CRISPR/Cas9 screens for new regulators of B cell responses. (A–E) Results of the CRISPR/Cas9 screens performed upon TD immunization. (A) Difference of sgRNA abundance in PBs versus donor B cells (left) and GC B cells versus donor B cells (right) is shown as log 2 FC for all sgRNAs in the library, neutral control sgRNAs targeting olfactory receptor genes ( Olfr ), sgRNAs targeting essential genes, and sgRNAs targeting the well-known PB regulators Irf4 and Prdm1 . (B) Volcano plot displaying the depletion or enrichment of sgRNAs (x axis) in PBs versus donor B cells (left) and in GC B cells versus donor B cells (right). The P values (y axis) were calculated using MAGeCK . For each gene, the sgRNA with the more significant P value was plotted. A twofold change in sgRNA abundance is indicated by a dashed line. Potentially positive (brown, P < 0.05, log 2 FC ≤ −1) and negative (blue, P < 0.05, log 2 FC ≥ 1) regulators are highlighted for PBs (left plot) and GC B cells (right plot). (C–E) Heat map showing the depletion or enrichment of sgRNAs in PBs versus control donor B cells (left column) and in GC B cells versus control donor B cells (right column). The different color shadings indicate the log 2 FC, while the circle size refers to the P value (−log 10 ). Genes uniquely found in our in vivo screens for PB regulators are highlighted in green color. Erlec1, Qpctl , Zfyve21 , and Isg20 were previously identified in an in vitro CRISPR/Cas9 screen as regulators of antibody secretion, but not as regulators of PB differentiation . The sgRNA depletion data for well-known positive regulators of plasma cell development (above) and positive regulators involved in the homeostasis of the ER (below) are shown in C. The sgRNA depletion data of novel potentially positive regulators of PB differentiation or homeostasis are shown in D. The sgRNA enrichment data for novel potentially negative regulators of PB differentiation or homeostasis are shown in E. ns, nonsignificant. (F–H) Results of the CRISPR/Cas9 screens performed upon TI immunization. (F) Volcano plot displaying the depletion or enrichment of sgRNAs (x axis) in PBs versus donor B cells (left) and memory B cells (Mem B, TACI + CD138 − ) versus donor B cells (right). The P values (y axis) were calculated using MAGeCK. For each gene, the sgRNA with the more significant P value was plotted. Potentially positive (brown, P < 0.05 and log 2 FC ≤ −1) and negative (blue, P < 0.05 and log 2 FC ≥ 1) regulators are highlighted for PBs (left) and memory B cells (right). (G and H) Common potential PB regulators identified in the TI and TD CRISPR/Cas9 screening experiments. The heat maps show the depletion (G) or enrichment (H) of the sgRNAs, which were identified in the TI CRISPR/Cas9 screen by comparing PBs versus control donor B cells (left column) and memory B cells versus control donor B cells (right column). The sgRNA depletion data for well-known positive regulators of plasma cell development (left) and novel potentially positive regulators of PB differentiation or homeostasis are shown in G, while the respective data for potentially negative regulators are indicated in H. The Tnfrsf17 (BCMA) sgRNA was depleted only in the TI sgRNA screen. ns, nonsignificant. Multiple replicate samples were analyzed in two independent sgRNA screening experiments in response to TD or TI immunization, as described in detail in (TD) and (TI). FC, fold change; PB, plasmablast.

Article Snippet: CD43 – B cells were enriched from the spleen or lymph nodes of mice by immunomagnetic depletion of non-B cells using CD43 (Ly-48) MicroBeads (Miltenyi Biotec).

Techniques: CRISPR, Control, Olfactory, In Vivo, In Vitro, Clinical Proteomics

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: CRISPR/Cas9 screen for identifying new regulators of the TI B cell responses. (A) Schematic diagram of the in vivo CRISPR/Cas9 screening experiments used to study the TI B cell responses. The TI screening experiment was performed as described in detail for the TD screening experiment , except that the transplanted mice were immunized with NP-Ficoll and that plasmablasts (TACI + CD138 + ) and memory B cells (Mem B, TACI + CD138 − ) were isolated at day 6 or 7 by flow cytometry. (B) Flow cytometric analysis of enriched mCherry + CD45.2 + B cells from recipient mice. The gates used for the sorting of plasmablasts and memory B cells are indicated. The percentages of mCherry + plasmablasts and mCherry + memory B cells within the sorted cell population are shown. (C) Numbers of sorted plasmablasts and memory B cells (left), the percentage of mCherry + plasmablasts and mCherry + memory B cells within the sorted cell population (middle), and the numbers of sorted mCherry + plasmablasts and mCherry + memory B cells (right) were determined for individual recipient mice analyzed in two independent screening experiments. (D) Total sgRNA representation in each plasmablast and memory B cell replicate sample analyzed in two screening experiments. The sgRNA representation was estimated by dividing the number of mCherry + plasmablasts or mCherry + memory B cells by the number of the 882 sgRNAs constituting the sgRNA library. Each plasmablast and memory B cell replicate sample contained cells that were obtained from seven or nine recipient mice after pooling. Each colored box represents the individual contribution of each mouse to the sgRNA representation of the entire replicate sample. (E) Flow cytometric definition of mCherry + memory B cells. Sorted mCherry + donor B cells were transferred to recipient mice, which were immunized 16 h later with NP-Ficoll in PBS. Flow cytometric analysis of splenocytes 6 days after immunization is shown. Donor-derived mCherry + cells were mainly TACI + CD138 + plasmablasts or CD138 − GL7 − CD38 + TACI + cells that phenotypically correspond to memory B cells.

Article Snippet: CD43 – B cells were enriched from the spleen or lymph nodes of mice by immunomagnetic depletion of non-B cells using CD43 (Ly-48) MicroBeads (Miltenyi Biotec).

Techniques: CRISPR, In Vivo, Isolation, Flow Cytometry, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: Validation of gene hits of the TD B cell responses by sgRNA-mediated gene inactivation. (A) Schematic diagram of the experiments used for validating potential regulators identified by the in vivo CRISPR/Cas9 screens. Naïve splenic B cells of the donor genotype were transduced with LV particles expressing the mCherry reporter protein and a sgRNA targeting the gene of interest. In parallel, donor B cells were transduced with LV particles expressing the mAmetrine reporter protein and the control sgRNA (sg. Chr1 ). After infection, the cells were cultured in vitro for 3 days in the presence of OP9 cells and sBAFF. Transduced mAmetrine + and mCherry + B cells were sorted by flow cytometry, mixed, and transferred to recipient mice, which were then immunized with NP-KLH/alum 16 h later. The frequency of mAmetrine + and mCherry + PBs in the spleen was analyzed by flow cytometry 7 days after immunization. (B) mCherry + and mAmetrine + sgRNA-expressing B cells were mixed at a 2.5:1 ratio and transferred into recipient mice, followed by immunization. Flow cytometric analysis of splenocytes from recipient mice was performed 7 days after immunization. mCherry + B cells expressed either the control sgRNA (sg. Chr1 ) or sgRNAs targeting Prdm1 (sg. Prdm1 ), positive regulator genes (sg. Amigo2 , sg. Slc22a17 , and sg. Qpctl ), and a negative regulator gene (sg. Cd44 ). The percentage of mCherry + (red gate) and mAmetrine + (blue gate) B cells among the total B cells (CD19 + CD138 –/lo and CD19 lo CD138 + ; green gate) is shown. The percentages of mCherry + and mAmetrine + PBs (TACI + CD138 + ) and GC B cells (GL7 + CD95 + ) in total B cells (green numbers) and within the mCherry + B cells (red numbers) or mAmetrine + B cells (blue numbers) are indicated. PB, plasmablast.

Article Snippet: CD43 – B cells were enriched from the spleen or lymph nodes of mice by immunomagnetic depletion of non-B cells using CD43 (Ly-48) MicroBeads (Miltenyi Biotec).

Techniques: Biomarker Discovery, In Vivo, CRISPR, Transduction, Expressing, Control, Infection, Cell Culture, In Vitro, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: Validation of positive and negative regulators of plasmablast and GC B cell formation by sgRNA-mediated gene inactivation. (A) mCherry + and mAmetrine + sgRNA-expressing B cells were mixed at a 2.5:1 ratio and transferred into recipient mice, followed by NP-KLH immunization. Flow cytometric analysis of splenocytes from recipient mice was performed 7 days after immunization. mCherry + B cells expressed either the control sgRNA (sg. Chr1 ) or sgRNAs targeting Prdm1 (sg. Prdm1 ), positive regulator genes (sg. Manf and sgZyve21 ), or negative regulator genes (sg. Prdx4 and sg. Atxn1 ). The percentage of mCherry + (red gate) and mAmetrine + (blue gate) B cells among the total B cells (CD19 + CD138 –/lo and CD19 lo CD138 + ; green gate) is shown. The percentages of mCherry + and mAmetrine + plasmablasts (TACI + CD138 + ) and GC B cells (GL7 + CD95 + ) in total B cells (green numbers) and within the mCherry + B cells (red numbers) or in mAmetrine + B cells (blue numbers) are indicated. (B and C) Analysis of the loss (left) or gain (right) of plasmablasts and GC B cells upon sgRNA-mediated inactivation of candidate genes coding for positive or negative regulators, respectively. The ratio of the percentage of mCherry + plasmablasts (B) or mCherry + GC B cells (C) (expressing the sg.Control [sg. Chr1 ] or sgRNAs targeting the indicated genes) versus mAmetrine + plasmablasts (B) or mAmetrine + GC B cells (C) (expressing the sg.Control) was determined in total B cells, respectively. The calculation of the different ratios is explained in detail in . The ratios, which were determined by analyzing different control mice (gray), experimental mice (black), and mice with inactivation of the known regulator genes Prdm1 or Irf4 (red), are indicated. Statistical data are shown as mean values with SEM and were analyzed by the multiple unpaired t test with Holm–Šídák’s multiple comparisons test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Each dot represents one mouse. The validation experiments were performed at least two times.

Article Snippet: CD43 – B cells were enriched from the spleen or lymph nodes of mice by immunomagnetic depletion of non-B cells using CD43 (Ly-48) MicroBeads (Miltenyi Biotec).

Techniques: Biomarker Discovery, Expressing, Control

Journal: The Journal of Experimental Medicine

Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation

doi: 10.1084/jem.20250594

Figure Lengend Snippet: Validation of the indicated regulators of TD B cell responses by sgRNA-mediated gene inactivation. (A and B) Flow cytometric analysis of two independent validation experiments performed by sgRNA-mediated inactivation of putative positive and negative regulators. The outline of the strategy and flow cytometric analysis of the validation experiments is explained in detail in . mCherry + and mAmetrine + sgRNA-transduced B cells were mixed at a ratio of 2.5:1 and transferred into recipient mice, followed by immunization with NP-KLH/alum. Flow cytometric analysis of splenocytes from recipient mice was performed 7 days after immunization. mCherry + donor B cells expressed the control sgRNA (sg. Chr1 ) or sgRNAs targeting positive regulator genes (sg. Isg20 , sg. Ccrl2 , sg. Tmem198 , and sg. Aldh1l2 ) or negative regulator genes (sg. Prg3 and sg. Stk3 ), while the mAmetrine + donor B cells expressed only the control sgRNA. The percentages of mCherry + and mAmetrine + plasmablasts (TACI + CD138 + ) and GC B cells (GL7 + CD95 + ) in total B cells (CD19 + CD138 –/lo and CD19 lo CD138 + ; green), in mCherry + cells (red), or in mAmetrine + cells (blue) are shown. The ratios of mCherry + versus mAmetrine + plasmablasts and GC B cells were quantified and calculated, as explained in C, and are shown in . (C) Calculations of the ratio of mCherry + to mAmetrine + plasmablasts in total B cells relative to the control. The corresponding results are shown in . First, the ratio (R) of the percentage of mCherry + plasmablasts (sg.Control [sg. Chr1 ]) in total B cells versus the percentage of mAmetrine + plasmablasts (sg.Control) in total B cells was determined for each control (c) mouse. Subsequently, the mean value of the control group (mean control group) was determined by dividing the sum of all Rc values by the total number of control mice analyzed. In parallel, the ratio (R) of the percentage of mCherry + plasmablasts (for each sgRNA test gene) in total B cells versus the percentage of mAmetrine + plasmablasts (sg.Control) in total B cells was determined for each experimental (e) mouse. We then normalized the Re value of each experimental mouse by dividing it by the mean value of the control group, which allowed to plot all Re values of the different mice in the same graph by setting the mean Rc value to 1 as shown in . The same calculation was used to determine the ratios for GC B cells .

Article Snippet: CD43 – B cells were enriched from the spleen or lymph nodes of mice by immunomagnetic depletion of non-B cells using CD43 (Ly-48) MicroBeads (Miltenyi Biotec).

Techniques: Biomarker Discovery, Control