Journal: Scientific Reports

Article Title: Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells

doi: 10.1038/s41598-017-01627-1

Figure Lengend Snippet: Cell surface antigen analysis by two-color flow cytometry. ( a ) Phenotype analysis of lymphocytes of the lymph node by flow cytometric analysis with a combination of anti-CD3/-CD20 and anti-CD27/-CD20 antibodies. B cells are surrounded by a dotted line. ( b ) Phenotype analysis of B cells purified using CD19-microbeads by flow cytomeric analysis with a combination of anti-CD3/-CD20 antibodies. Purified B cells are surrounded by a dotted line.

Article Snippet: B cells were purified using magnetic-activated cell sorting CD19 Micro Beads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Purification

Journal: Scientific Reports

Article Title: Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells

doi: 10.1038/s41598-017-01627-1

Figure Lengend Snippet: Characterization of the BiPSCs. ( a ) Immunofluorescence staining of BiPSC13 and MIB2-6 for expression of the pluripotent markers Oct3/4, Nanog, SSEA4, TRA-1-60, and TRA-1-81. ( b ) Expression of endogenous Oct3/4, Sox2, Klf4, cMyc, Pax5, AID , and GAPDH in BiPSCs (BiPSC13, MIB2-6) and normal B cells (CD19) from the lymph node analyzed using RT-PCR. ( c ) RT-PCR analysis of the expression of retrovirus-derived Oct3/4, Sox2, Klf4 , and cMyc in BiPSCs (BiPSC13, MIB2-6). HUC-Fm, human umbilical cord fibroblast cells, (male; RIKEN, Tsukuba, Japan) infected with a retrovirus containing Oct3/4 , Sox2 , Klf4 , c-Myc , and GAPDH for 5 days were used as the positive control.

Article Snippet: B cells were purified using magnetic-activated cell sorting CD19 Micro Beads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Infection, Positive Control

Journal: Scientific Reports

Article Title: Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells

doi: 10.1038/s41598-017-01627-1

Figure Lengend Snippet: AID expression in BiPSCs induced with the doxycycline-controlled (Tet-off) system. ( a ) Induction of AID expression in the absence of doxycycline was confirmed in two clones (#1 and #2) derived from BiPSC13 by western blotting, and ( b ) qRT-PCR. In ( b ), the numbers on the Y axis are the relative ratio comparing the expression of AID mRNA of each sample standardized by the expression of GAPDH mRNA with that of CD19 + normal B cells purified from normal LN using CD19-microbeads. Raji, Burkitt lymphoma cell line. Data were analyzed in triplicates and normalized to glyceraldehyde 3-phosphate dehydrogenase. ( c ) Immunofluorescence analysis of the expression of Oct3/4 and Nanog in BiPSC13-AID (#1 and #2) after the induction of AID expression in the absence of doxycycline. ( d ) Induction of AID expression in the absence of doxycycline in two clones (#16 and #17) derived from MIB2-6 by western blotting, and ( e ) qRT-PCR as described in the legend to ( b ). ( f ) Immunofluorescence analysis of the expression of Oct3/4 and Nanog in MIB-AID (#16 and #17) after the induction of AID expression in the absence of doxycycline.

Article Snippet: B cells were purified using magnetic-activated cell sorting CD19 Micro Beads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Clone Assay, Derivative Assay, Western Blot, Quantitative RT-PCR, Purification, Immunofluorescence

Journal: Scientific Reports

Article Title: STAT3 deficiency in B cells exacerbates uveitis by promoting expansion of pathogenic lymphocytes and suppressing regulatory B cells (Bregs) and Tregs

doi: 10.1038/s41598-020-73093-1

Figure Lengend Snippet: Generation and characterization of STAT3 conditional KO mice. Stat 3 fl/fl mice were crossed with CD19-Cre mice to generate mice with deletion of stat3 in CD19 + B cells (CD19-STAT3KO). ( A ) CD19-STAT3KO mice were identified by PCR analysis of mouse tail genomic DNA. ( B ) Naive CD19 + B cells from LN and spleens of control (CD19 +/CRE STAT3 f/f ) or CD19-STAT3KO mice were stimulated with LPS and analyzed by Western blotting. ( C ) Splenocytes were isolated from control or CD19-STAT3KO mice and cells were counted and immediately used for surface FACS analysis with anti-CD4 or anti-CD19 mAbs after dead cell exclusion. Numbers in the quadrants are the percentage of CD4 + T cells or CD19 + B cells. Histograms presented in ( C ) represent the absolute number of T cells or B cells per mouse (n = 6). Results represent at least 3 independent studies. * p < 0.05, ** p < 0.01, *** p < 0.001, N.S (Student two-tailed t test).

Article Snippet: Primary CD19 + B-cells (> 96%) were isolated from the spleen/LN of wild type control and CD19-STAT3KO mice by using anti-CD19 magnetic sorting beads (order number: 130-052-201, Miltenyi Biotec Inc. CA).

Techniques: Western Blot, Isolation, Two Tailed Test

Journal: Scientific Reports

Article Title: STAT3 deficiency in B cells exacerbates uveitis by promoting expansion of pathogenic lymphocytes and suppressing regulatory B cells (Bregs) and Tregs

doi: 10.1038/s41598-020-73093-1

Figure Lengend Snippet: STAT3 regulates B cell proliferation and STAT3-deficient B cells exhibit a pre-activated phenotype. ( A – C ) CD19 + B cells isolated from control or CD19-STAT3KO mouse spleen were activated with LPS and subjected to thymidine incorporation assay ( A ), CFSE dilution assay ( B ) or DNA analysis by the propidium iodine assay ( C ). ( D ) Freshly isolated B cells from the spleen were stained with Abs specific to cell surface proteins indicated and analyzed by FACS. Graphs show relative amounts of the CD19 + B cells expressing the various cell surface markers (mean and SEM, n = 6). ( E ) Naive CD19 + B cells from spleen of control and CD19-STAT3KO mice were analyzed by qPCR. ( F ) Purified CD19 + B cells from naïve control or CD19-STAT3KO mouse spleen were activated with anti-CD40 and anti-IgM antibody for 48 h and glycolic assays were performed using the Seahorse Glycolytic Rate XFp kit (Agilent Technology, Cedar Creek, TX). Results are presented as Proton Production Rate (PPR). ( G ) Apoptosis assays were performed using CD19-STAT3KO or control B cells activated with anti-CD40 and anti-IgM antibodies for 48 h. ( H ) RT-PCR analysis of RNA obtained from the activated B cells and bands detected by gel electrophoresis were quantified by qPCR (right panels). Results represent at least 3 independent studies. * p < 0.05, ** p < 0.01, *** p < 0.001, N.S (Student two-tailed t test).

Article Snippet: Primary CD19 + B-cells (> 96%) were isolated from the spleen/LN of wild type control and CD19-STAT3KO mice by using anti-CD19 magnetic sorting beads (order number: 130-052-201, Miltenyi Biotec Inc. CA).

Techniques: Isolation, Thymidine Incorporation Assay, Dilution Assay, Staining, Expressing, Purification, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Two Tailed Test

Journal: Scientific Reports

Article Title: STAT3 deficiency in B cells exacerbates uveitis by promoting expansion of pathogenic lymphocytes and suppressing regulatory B cells (Bregs) and Tregs

doi: 10.1038/s41598-020-73093-1

Figure Lengend Snippet: CD19-STAT3KO mice developed severe EAU. EAU was induced in control or CD19-STAT3KO mice by immunization with the uveitogenic peptide, IRBP 651-670 in CFA and disease progression was analyzed by fundoscopy, histology, optical coherence tomography (OCT) and electroretinography (ERG). ( A ) Fundus image of retina at day 21 after EAU induction were taken using an otoendoscopic imaging system. Fundus images reveal inflammation with blurred optic disc margins and enlarged juxtapupillary area (black arrows), retinal vasculitis (blue arrows) and yellow-whitish retinal and choroidal infiltrates (white arrows). OpN, optic nerve. Clinical scores and assessment of disease severity were based on changes at the optic nerve disc or retinal vessels and retinal and choroidal infiltrates. ( B ) Histologic images of the eyes were from eyes harvested 21 days after EAU immunization and show substantial numbers of inflammatory cells in the CD19-STAT3 retina. H&E histological sections: Scale bar, 100 µM. V, vitreous; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE/CH retinal pigmented epithelial and choroid. Blue arrows, lymphocytes; Asterisks, retinal-folds. Histogram to the right shows the clinical scores, N = 10. ( C ) Representative OCT images show marked increase of inflammatory cells (white arrows) in the vitreous of mice and disturbance of the retinal layer structure with granulomatous lesions (red arrows) were more pronounced in the CD19-STAT3KO retina. ( D ) ERG analysis of the retina on day-20 after EAU induction. The averages of light- or dark-adapted ERG a-wave or b-wave amplitudes are plotted as a function of flash luminance and values are means ± SEM from 4 animals in each group. Data are presented as the mean ± SEM of at least three determinations. Results represent 3 independent studies. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary CD19 + B-cells (> 96%) were isolated from the spleen/LN of wild type control and CD19-STAT3KO mice by using anti-CD19 magnetic sorting beads (order number: 130-052-201, Miltenyi Biotec Inc. CA).

Techniques: Tomography, Imaging

Journal: Scientific Reports

Article Title: STAT3 deficiency in B cells exacerbates uveitis by promoting expansion of pathogenic lymphocytes and suppressing regulatory B cells (Bregs) and Tregs

doi: 10.1038/s41598-020-73093-1

Figure Lengend Snippet: Exacerbated EAU in CD19-STAT3KO mice correlate with expansion of Th17 cells. ( A ) Lymphocytes were isolated from control or CD19-STAT3KO EAU mice and proliferative responses of the cells to IRBP 651-670 were assessed by 3 H-thymidine incorporation assay. Five replicate cultures were analyzed, and data presented as mean value of CPM of the five replicate cultures. ( B , C ) PBMC from peripheral blood of control or CD19-STAT3KO EAU mice were stained and analyzed by intracellular cytokine staining assay and FACS. Numbers in quadrants indicate percentage of T cells expressing IL-17 and/or IFN-γ. Data are presented as mean ± SEM of three replicates. Results represent 3 independent studies. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary CD19 + B-cells (> 96%) were isolated from the spleen/LN of wild type control and CD19-STAT3KO mice by using anti-CD19 magnetic sorting beads (order number: 130-052-201, Miltenyi Biotec Inc. CA).

Techniques: Isolation, Thymidine Incorporation Assay, Staining, Expressing

Journal: Scientific Reports

Article Title: STAT3 deficiency in B cells exacerbates uveitis by promoting expansion of pathogenic lymphocytes and suppressing regulatory B cells (Bregs) and Tregs

doi: 10.1038/s41598-020-73093-1

Figure Lengend Snippet: CD19-STAT3KO mice are defective in generating Breg and Treg cells during EAU. ( A ) Freshly isolated CD4 + T cells in the dLNs and spleen of EAU mice were re-stimulated with IRBP and counted using the Vi-Cell XR cell viability analyzer (Beckman Coulter). ( B ) Freshly isolated PBMC cells from control or CD19-STAT3KO EAU mice were gated by FACS for CD11b + , CD4 + , and CD19 + cells after dead cell exclusion. ( C ) CD4 + T cells or ( D ) CD19 + B cells expressing IL-10 or IL-35 (p35 and EBI3) were detected and quantified by the intracellular cytokine staining assays. Numbers in quadrants and the corresponding histograms indicate percentage of IL-10-, p35- and/or Ebi3-expressing cells. ( E ) RNA isolated from the CD19 + B cells were analyzed by qPCR. The data are presented as the mean ± SEM of at least three determinations. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary CD19 + B-cells (> 96%) were isolated from the spleen/LN of wild type control and CD19-STAT3KO mice by using anti-CD19 magnetic sorting beads (order number: 130-052-201, Miltenyi Biotec Inc. CA).

Techniques: Isolation, Expressing, Staining

Journal: Scientific Reports

Article Title: STAT3 deficiency in B cells exacerbates uveitis by promoting expansion of pathogenic lymphocytes and suppressing regulatory B cells (Bregs) and Tregs

doi: 10.1038/s41598-020-73093-1

Figure Lengend Snippet: Upregulation of CD80 anf CD86 and downregulation of Lag3 on CD19-STAT3KO B cells during EAU. A Draining LN and splenocytes from control or CD19-STAT3KO EAU mice were gated by FACS analysis into CD4 + , CD19 + cells after dead cell exclusion. B Numbers in quadrants A and corresponding histograms indicate percentage of CD19 + cells expressing costimulatory molecules (CD80, CD86) or LAG3. ( C ) CD19 + B-cells from control or CD19-STAT3KO EAU mice were co-cultured with B cell-depleted dLN cells from control EAU mice (1:1) for 3-days in medium containing IRBP 651-670 . Intracellular cytokine analysis was performed and numbers in quadrants and histograms indicate percentages of IL-17- and IFN-γ-expressing CD4 + T cells. The data are presented as the mean ± SEM of at least three determinations. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary CD19 + B-cells (> 96%) were isolated from the spleen/LN of wild type control and CD19-STAT3KO mice by using anti-CD19 magnetic sorting beads (order number: 130-052-201, Miltenyi Biotec Inc. CA).

Techniques: Expressing, Cell Culture

Journal: Scientific Reports

Article Title: STAT3 deficiency in B cells exacerbates uveitis by promoting expansion of pathogenic lymphocytes and suppressing regulatory B cells (Bregs) and Tregs

doi: 10.1038/s41598-020-73093-1

Figure Lengend Snippet: CD19-STAT3KO mice developed severe EAE. ( A ) EAE was induced in CD19-STAT3KO (N = 4) or WT control mice (N = 4) by immunization with MOG 35-55 -peptide in CFA. Clinical disease scores were assessed daily, from day 8 until day 16, by masked investigators. ( B ) Infiltrated leukocytes were isolated from spinal cord and brain. Viable CD45 + CD4 + or CD45 + CD19 + cell numbers from spinal cord and brain were determined by FACS analysis. ( C , D ) The cells were subjected to intracellular cytokine staining to detect percentage of cytokine expression in CD4 + T cell ( C ) and CD19 + B cell compartments ( D ). ( E ) Draining LN cells were isolated and counted, and analyzed for cell surface expression of CD4 or CD19 by FACS. ( F ) The draining LN cells were stimulated with MOG 35-55 -peptide for 48 h and subjected to 3 H-thymidine incorporation assay. Data are presented as mean CPM ± S.E.M. of responses of 4 replicate cultures. Data represents > 3 independent experiments (** P < 0.01; *** P < 0.001; **** P < 0.0001).

Article Snippet: Primary CD19 + B-cells (> 96%) were isolated from the spleen/LN of wild type control and CD19-STAT3KO mice by using anti-CD19 magnetic sorting beads (order number: 130-052-201, Miltenyi Biotec Inc. CA).

Techniques: Isolation, Staining, Expressing, Thymidine Incorporation Assay

Journal: Journal of Virology

Article Title: A New Model of Epstein-Barr Virus Infection Reveals an Important Role for Early Lytic Viral Protein Expression in the Development of Lymphomas ▿

doi: 10.1128/JVI.01512-10

Figure Lengend Snippet: Reconstitution of human hematopoietic cells in NSG mice. Peripheral blood was collected from immune reconstituted hNSG(thy) mice 10 weeks after human fetal CD34 cell transplantation and stained with antibodies specific for human CD45, CD3, and CD19 by flow cytometry. Dead cells were excluded from the analysis. The reconstitution levels in 12 mice that were subsequently infected with control EBV, versus 16 mice that were subsequently infected with Z-KO EBV, are shown. Results are presented as the percentages of positively staining cells with each antibody in comparison to the total leukocyte population.

Article Snippet: The splenocytes were depleted of B cells by magnetic sorting using anti-CD19 beads (Miltenyi Biotech) and labeled with 2.5 μM carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, Eugene, OR).

Techniques: Transplantation Assay, Staining, Flow Cytometry, Infection, Comparison

Journal: STAR Protocols

Article Title: Protocol for correlation analysis of the murine gut microbiome and meta-metabolome using 16S rDNA sequencing and UPLC-MS

doi: 10.1016/j.xpro.2022.101494

Figure Lengend Snippet:

Article Snippet: Remove CD3 + T cells by magnetic bead sorting (CD3 Selection Kit, Miltenyi Biotec). c. Purify splenic T cells by negative sorting using magnetic microbeads from the donor’s spleen (Pan T-Cell Isolation Kit, Miltenyi Biotec). d. Deliver 5×10 6 T-cell-depleted bone marrow (TCD-BM) cells with or without 5×10 5 splenic T cells to recipients on day 0 after lethal irradiation, while T cells are used as a source for aGVHD induction. e. Assess the animals for weight loss, clinical condition and disease progression twice a week according to the criteria of the disease model.

Techniques: Cell Isolation, Isolation, Control, Modification, Amplification, Sequencing, Software, Recombinant, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: Phorbol-12-myristate-13-acetate is a potent enhancer of B cells with a granzyme B + regulatory phenotype

doi: 10.3389/fimmu.2023.1194880

Figure Lengend Snippet: Differentiation of pre-activated B cells into either plasma cells or GrB + regulatory GraB cells and concept of generation and application of GrB + regulatory B cells for the treatment of hyper-inflammatory diseases. (A) Regular CD4 + T cell activation includes stimulation of both the T cell receptor (TCR) via MHC/peptide complexes and CD28 via B7. Such fully activated T cells secrete IL-21 and express high levels of CD40 ligand (CD40L), enabling them to induce plasma cell differentiation in B cells, when they receive antigen-specific signals via their B cell receptor (BCR) in parallel. In contrast, in certain pathological situations such as during an HIV infection, the TCR of CD4 + T cells can be directly stimulated (e.g. via the protein Nef during HIV infection), without simultaneous costimulation of CD28. Such incompletely activated T cells secrete IL-21, but barely express CD40L, resulting in the induction of GrB-secreting GraB cells instead of plasma cells. Experimentally, exogenous addition of CD40L multimers can substitute for incomplete B cell/T cell interactions, resulting in enhanced plasma cell differentiation in vitro . (B) GrB + regulatory B cells ( GraB cells ) can be generated from either autologous or allogeneic, HLA-matched donor blood. After autologous or allogeneic leukapheresis B cells are enriched, for example by magnetic depletion of CD3 + T cells, followed by positive magnetic selection of CD19 + cells using MACS technology. Subsequently, B cells are cultured in a clean room environment (A-in-B clean room environment) in the presence of IL-21, the key cytokine for the generation of GraB cells . In order to achieve robust stimulation of alloreactive pre-activated B cells, a second stimulus is required, which can either directly target the B cell receptor or subordinate signaling pathways downstream of the B cell receptor as discussed later. After an incubation period of 48 hours, alloreactive pre-activated B cells express GrB and can be harvested from the incubation flasks. After several washing steps to remove remnants of stimulatory agents in the medium, cell suspensions are quality-tested to ensure purity, absence of endotoxin and microbiological contamination including mycoplasma. Then, the final cell product can be released and either cryopreserved or directly transfused to the patient. Potential indications are hyper-inflammatory diseases including autoimmune or graft-versus-host diseases. Anti-BCR, antibodies directed against the constant part of the B Cell Receptor; B7, Costimulatory molecules CD80 and CD86; BCR, B Cell Receptor; CD, Cluster of Differentiation; CD40L, CD40 Ligand; GvHD, Graft-versus-Host Disease; HLA, Human Leukocyte Antigen; IL-21, Interleukin 21; IL-21R, IL-21 Receptor; MACS, Magnetic Activated Cell Sorting; MHC, Major Histocompatibility Complex; PMA, Phorbol Myristate Acetate; TCR, T Cell Receptor.

Article Snippet: Subsequently, CD19 + B cells were positively selected by Magnetic Activated Cell Sorting according to the manufacturer´s instructions (CliniMACS CD19, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activation Assay, Cell Differentiation, Infection, In Vitro, Generated, Selection, Cell Culture, Incubation, FACS

Journal: Frontiers in Immunology

Article Title: Phorbol-12-myristate-13-acetate is a potent enhancer of B cells with a granzyme B + regulatory phenotype

doi: 10.3389/fimmu.2023.1194880

Figure Lengend Snippet: IL-21 induces and antibodies to the B cell receptor strongly enhance differentiation of pre-activated B cells into GrB + GraB cells. B cells from healthy donors were isolated from PBMC using magnetic beads directed against CD19. Subsequently, B cells were incubated for 48 hours in the presence of increasing concentrations of IL-21 and anti-BCR as indicated. Then, B cells were harvested, stained for intracellular GrB and analyzed by flow cytometry. (A) Dot plots show percentages of GrB + GraB cells from one representative experiment out of n > 10. (B, C) Box plots show average percentages of GrB + GraB cells from n > 10 individual experiments as indicated, each dot indicates a result from one individual experiment. Box central horizontal lines indicate medians, box borders represent IQR, whiskers indicate minima and maxima. Significance levels were *** p < 0.0005, ** p < 0.005 and * p < 0.05. Anti-BCR, activating antibodies against the constant part of the B Cell Receptor; GrB, Granzyme B; IgG, Immunoglobulin G; IL-21, Interleukin 21; IQR, interquartile ranges; ns, not significant; PBMC, Peripheral Blood Mononuclear Cells; PE, Phycoerythrin; SSC, Side Scatter.

Article Snippet: Subsequently, CD19 + B cells were positively selected by Magnetic Activated Cell Sorting according to the manufacturer´s instructions (CliniMACS CD19, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Isolation, Magnetic Beads, Incubation, Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Phorbol-12-myristate-13-acetate is a potent enhancer of B cells with a granzyme B + regulatory phenotype

doi: 10.3389/fimmu.2023.1194880

Figure Lengend Snippet: Optimal doses of PMA differentiate pre-activated B cells into GrB + GraB cells and improve their viability ex vivo. B cells from at least ten healthy donors were isolated from PBMC using magnetic beads directed against CD19. Subsequently, B cells were incubated for 48 hours in the presence of increasing concentrations of IL-21 and PMA as indicated. Anti-BCR at 12 µg/ml and IL-21 at 100 ng/ml served as positive control. Then, B cells were harvested, stained for intracellular GrB and analyzed by flow cytometry. (A) Line graphs show mean percentages of GrB + GraB cells from n > 10 individual experiments as indicated. (B) Line graphs show mean viability of GraB cells at the end of the cell culture period based on morphological criteria. Error bars indicate SEM. Anti-BCR, activating antibodies against the constant part of the B Cell Receptor; B21, Anti-BCR + IL-21; GrB, Granzyme B; IL-21, Interleukin 21; PMA, Phorbol Myristate Acetate; SEM, Standard Error of the Mean.

Article Snippet: Subsequently, CD19 + B cells were positively selected by Magnetic Activated Cell Sorting according to the manufacturer´s instructions (CliniMACS CD19, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Ex Vivo, Isolation, Magnetic Beads, Incubation, Positive Control, Staining, Flow Cytometry, Cell Culture

Journal: Frontiers in Immunology

Article Title: Phorbol-12-myristate-13-acetate is a potent enhancer of B cells with a granzyme B + regulatory phenotype

doi: 10.3389/fimmu.2023.1194880

Figure Lengend Snippet: PMA may replace activating antibodies against the B cell receptor to enhance IL-21-dependent ex-vivo differentiation of pre-activated B cells into GrB + GraB cells. B cells were isolated from PBMC using magnetic beads directed against CD19. Subsequently, B cells were incubated for 48 hours in the presence of different combinations of IL-21 at 100 ng/ml, anti-BCR at 12 µg/ml and PMA at 50 ng/ml as indicated. Then, B cells were harvested, stained for intracellular GrB and analyzed by flow cytometry. (A) Dot plots show percentages of GrB + GraB cells from one representative experiment out of n > 10. (B) Box plots show average percentages of GrB + GraB cells from n > 10 individual experiments as indicated. Box central horizontal lines indicate medians, box borders represent IQR, whiskers indicate minima and maxima. Significance level was * p < 0.05. Anti-BCR, activating antibodies against the constant part of the B Cell Receptor; B21, Anti-BCR + IL-21; GrB, Granzyme B; IL-21, Interleukin 21; IQR, interquartile ranges; ns, not significant; P21, PMA + IL-21; PBMC, Peripheral Blood Mononuclear Cells; PE, Phycoerythrin; PMA, Phorbol Myristate Acetate; SSC, Side Scatter.

Article Snippet: Subsequently, CD19 + B cells were positively selected by Magnetic Activated Cell Sorting according to the manufacturer´s instructions (CliniMACS CD19, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Ex Vivo, Isolation, Magnetic Beads, Incubation, Staining, Flow Cytometry