bcl6 pe  (Miltenyi Biotec)

Journal: Cell reports

Article Title: Ipsilateral immunization after a prior SARS-CoV-2 mRNA vaccination elicits superior B cell responses compared to contralateral immunization

doi: 10.1016/j.celrep.2023.113665

Figure Lengend Snippet: (A) Schematic illustration of the experimental design. Mice were primed with 0.2 μg SARS-CoV-2 mRNA vaccine and boosted 3 weeks later either in ipsilateral or contralateral limb. Mice that did not receive booster vaccination were used for comparison. Ipsilateral dLNs to the primary vaccination were analyzed by flow cytometry 4 days post boost. (B) Groups were compared for the numbers of total live cells, B cells, and GCBCs. (C) Flow cytometry analysis of c-MYC expression in total and RBD-specific GCBCs. (D) Flow cytometry analysis of plasma cell precursors (BCL6 lo IFR4 hi ) within total and RBD-specific GCBC populations. (E) Total plasma blasts and RBD-specific plasma blasts were analyzed by flow cytometry. For (B) and (C), n = 9–10 from three independent experiments. For (D) and (E). n = 6–7 from two independent experiments. Each dot represents one mouse, with lines depicting the median. All data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: not significant. Also see for day 2 analyses.

Article Snippet: The cells were then stained with anti-IRF4 (Alexa Fluor 647, BioLegend Cat: 646408), anti-BCL6 (PE-CF594, BD Horizon Cat: 562401) antibodies, c-MYC (Cell Signaling Technology, cat: 18583S, conjugated by secondary antibody), anti-Rabbit 2 nd antibody (Cy3 conjugated, BioLegend Cat: 406402).

Techniques: Comparison, Flow Cytometry, Expressing

Journal: Cell reports

Article Title: Ipsilateral immunization after a prior SARS-CoV-2 mRNA vaccination elicits superior B cell responses compared to contralateral immunization

doi: 10.1016/j.celrep.2023.113665

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The cells were then stained with anti-IRF4 (Alexa Fluor 647, BioLegend Cat: 646408), anti-BCL6 (PE-CF594, BD Horizon Cat: 562401) antibodies, c-MYC (Cell Signaling Technology, cat: 18583S, conjugated by secondary antibody), anti-Rabbit 2 nd antibody (Cy3 conjugated, BioLegend Cat: 406402).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, In Vivo, Software

Journal: PLOS ONE

Article Title: Release from persistent T cell receptor engagement and blockade of aryl hydrocarbon receptor activity enhance IL-6-dependent mouse follicular helper T-like cell differentiation in vitro

doi: 10.1371/journal.pone.0287746

Figure Lengend Snippet: Naïve CD4 + T cells were stimulated with immobilized mAbs to CD3, ICOS, and LFA-1 (3, 1.5, and 1.5 μg/ml for coating, respectively) in the presence of soluble anti-CD28 mAb (1 μg/ml), IL-6 (20 ng/ml), and neutralizing mAbs to IL-2 (10 μg/ml) and IL-4 (10 μg/ml) with or without blocking mAbs to IL-2Rα (CD25; 10 μg/ml) and IL-2Rβ (CD122; 10 μg/ml) for 3 days in DMEM. Relative mRNA expression of ( A ) Bcl6 , ( B ) Rorc(γt) , and ( C ) Prdm1 was analyzed by real-time PCR. ( D ) Intracellular expression of BCL6 protein was analyzed by flow cytometry after fixation and permeabilization. Representative flow cytometry histograms are also shown. Data are presented as mean ± SD of quadruplicate or triplicate samples. Results shown are representative of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Intracellular staining was performed with anti-BCL6 (clone K112-91) PE and anti-RORγt (Q31-378) Alexa Fluor 647 (both from BD Biosciences) or with anti-IL-21 (clone FFA21) PE (eBioscience) and anti-IL-17A (clone TC11-18H10.1) APC (BioLegend) using Foxp3/Transcription Factor Staining Buffer Sets (eBioscience) according to the manufacturer’s instructions.

Techniques: Blocking Assay, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: PLOS ONE

Article Title: Release from persistent T cell receptor engagement and blockade of aryl hydrocarbon receptor activity enhance IL-6-dependent mouse follicular helper T-like cell differentiation in vitro

doi: 10.1371/journal.pone.0287746

Figure Lengend Snippet: Naïve CD4 + T cells were stimulated as described in the legend of in the presence of blocking mAbs to IL-2Rs for 3 days (“3d”). Aliquots of cells were transferred to a new well without Abs to CD3, ICOS, and LFA-1 receiving an equal volume of fresh DMEM supplemented with IL-6 (20 ng/ml), anti-IL-2 (10 μg/ml), and anti-IL-2Rs (10 μg/ml each) and were further cultured for 2 days (“3+2d”). Other aliquots of cells received an equal volume of fresh DMEM supplemented as above in the same well, and further cultured for 2 days (“5d”) as schematically shown in ( A ). After the culture, relative mRNA expression of ( B ) Bcl6 and ( C ) Rorc(γt) were analyzed by real-time PCR. Data are presented as mean ± SD of triplicate samples. Results shown are representative of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Intracellular staining was performed with anti-BCL6 (clone K112-91) PE and anti-RORγt (Q31-378) Alexa Fluor 647 (both from BD Biosciences) or with anti-IL-21 (clone FFA21) PE (eBioscience) and anti-IL-17A (clone TC11-18H10.1) APC (BioLegend) using Foxp3/Transcription Factor Staining Buffer Sets (eBioscience) according to the manufacturer’s instructions.

Techniques: Blocking Assay, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

Journal: PLOS ONE

Article Title: Release from persistent T cell receptor engagement and blockade of aryl hydrocarbon receptor activity enhance IL-6-dependent mouse follicular helper T-like cell differentiation in vitro

doi: 10.1371/journal.pone.0287746

Figure Lengend Snippet: Naïve CD4 + T cells were stimulated for 3 days as described in the legend of but in the presence of graded concentrations of TGF-β1 in DMEM (“3d”). Aliquots of cells were transferred to a new well without immobilized Abs, receiving an equal volume of fresh DMEM supplemented with IL-6, graded concentrations of TGF-β1, and blocking Abs to IL-2 signaling, and were further cultured for 2 days (“3+2d”). After the “3d” culture ( open bar ) and the “3+2d” culture ( closed bar ), relative mRNA expression of ( A ) Bcl6 and ( B ) Rorc(γt) was analyzed by real-time PCR. ( C ) Recovered cell number from the “3d” culture and the “3+2d” culture is shown. Stars indicate that the cell number in the culture with TGF-β1 was significantly different from that in the culture without adding TGF-β1. Data are presented as mean ± SD of quadruplicate samples. Results shown are representative of three independent experiments. ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Intracellular staining was performed with anti-BCL6 (clone K112-91) PE and anti-RORγt (Q31-378) Alexa Fluor 647 (both from BD Biosciences) or with anti-IL-21 (clone FFA21) PE (eBioscience) and anti-IL-17A (clone TC11-18H10.1) APC (BioLegend) using Foxp3/Transcription Factor Staining Buffer Sets (eBioscience) according to the manufacturer’s instructions.

Techniques: Blocking Assay, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

Journal: PLOS ONE

Article Title: Release from persistent T cell receptor engagement and blockade of aryl hydrocarbon receptor activity enhance IL-6-dependent mouse follicular helper T-like cell differentiation in vitro

doi: 10.1371/journal.pone.0287746

Figure Lengend Snippet: Naïve CD4 + T cells were stimulated as described in the legend of in the presence or absence of added TGF-β1 (1 ng/mL). After the culture, aliquots of cells were fixed and permeabilized, and their intracellular expression of ( A ) BCL6 protein and ( B ) RORγt protein was analyzed by flow cytometry, and their ΔMFI ratios are shown ( C ). ( D ) Forward scatter (FSC)-A intensities of the cells before fixation/permeabilization are shown. Data are presented as mean ± SD of triplicate or quadruplicate samples. Results shown are representative of three independent experiments. ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Intracellular staining was performed with anti-BCL6 (clone K112-91) PE and anti-RORγt (Q31-378) Alexa Fluor 647 (both from BD Biosciences) or with anti-IL-21 (clone FFA21) PE (eBioscience) and anti-IL-17A (clone TC11-18H10.1) APC (BioLegend) using Foxp3/Transcription Factor Staining Buffer Sets (eBioscience) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry

Journal: PLOS ONE

Article Title: Release from persistent T cell receptor engagement and blockade of aryl hydrocarbon receptor activity enhance IL-6-dependent mouse follicular helper T-like cell differentiation in vitro

doi: 10.1371/journal.pone.0287746

Figure Lengend Snippet: Tfh-like cells were induced from naïve CD4 + T cells by the “3+2d” culture with TGF-β1 (1 ng/ml) as described in the legend of but using either DMEM ( black closed bar ) or RPMI 1604 medium ( gray bar ). Th0, Th1 and Th17 cells were induced from naïve CD4 + T cells in the RPMI 1640 medium as described in Materials and Methods. ( A and B ) Representative flow cytometry density plots of intracellular expression of Bcl6 and RORγt in (A) DMEM culture and (B) RPMI 1640 culture. The quadrants are based on the isotype control Abs, and the numbers in each quadrant indicate the percentage of cells in the quadrant. Relative mRNA expression of ( C ) Rorc(γt) , ( D ) Bcl6 , ( F ) Cxcr5 , ( G ) Ahr , and ( H ) Cyp1a1 was analyzed by real-time PCR. Cell surface expression of I CXCR5 was analyzed by flow cytometry. Data are presented as mean ± SD of quadruplicate samples. ( I ) The indicated helper T cells were treated with mitomycin C and cultured with B cells (1:1 T/B ratio) for 7 days. Production levels of whole IgG, assessed by ELISA, are shown. Data are presented as mean ± SD of triplicate samples. Results shown are representative of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Intracellular staining was performed with anti-BCL6 (clone K112-91) PE and anti-RORγt (Q31-378) Alexa Fluor 647 (both from BD Biosciences) or with anti-IL-21 (clone FFA21) PE (eBioscience) and anti-IL-17A (clone TC11-18H10.1) APC (BioLegend) using Foxp3/Transcription Factor Staining Buffer Sets (eBioscience) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: Release from persistent T cell receptor engagement and blockade of aryl hydrocarbon receptor activity enhance IL-6-dependent mouse follicular helper T-like cell differentiation in vitro

doi: 10.1371/journal.pone.0287746

Figure Lengend Snippet: Tfh-like cells were induced from naïve CD4 + T cells in DMEM as described in the legend of (“3+2d”) in the presence of graded concentrations of CH-223191. Relative mRNA expression of ( A ) Rorc(γt) , ( B ) Cxcr5 , and ( D ) Bcl6 was analyzed by real-time PCR. Cell surface expression of ( C ) CXCR5, ( E ) PD-1, and ( F ) ICOS was analyzed by flow cytometry. Data are presented as mean ± SD of triplicate samples. Results shown are representative of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Intracellular staining was performed with anti-BCL6 (clone K112-91) PE and anti-RORγt (Q31-378) Alexa Fluor 647 (both from BD Biosciences) or with anti-IL-21 (clone FFA21) PE (eBioscience) and anti-IL-17A (clone TC11-18H10.1) APC (BioLegend) using Foxp3/Transcription Factor Staining Buffer Sets (eBioscience) according to the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: PLOS ONE

Article Title: Release from persistent T cell receptor engagement and blockade of aryl hydrocarbon receptor activity enhance IL-6-dependent mouse follicular helper T-like cell differentiation in vitro

doi: 10.1371/journal.pone.0287746

Figure Lengend Snippet: Naïve CD4 + T cells were cultured as described in the legend of (“3+2d”) in RPMI in the presence of CH-223191 (5 μM), FICZ (100 nM), ITE (1 μM), or vehicle control ( A — F ). Relative mRNA expression of ( A ) Cxcr5 , ( C ) Rorc(γt) , and ( D ) Bcl6 was analyzed by real-time PCR. Cell surface expression of ( B ) CXCR5, ( E ) PD-1, and ( F ) ICOS was analyzed by flow cytometry. Data are presented as mean ± SD of quadruplicate samples. Data are presented as mean ± SD of triplicate samples. Results shown are representative of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Intracellular staining was performed with anti-BCL6 (clone K112-91) PE and anti-RORγt (Q31-378) Alexa Fluor 647 (both from BD Biosciences) or with anti-IL-21 (clone FFA21) PE (eBioscience) and anti-IL-17A (clone TC11-18H10.1) APC (BioLegend) using Foxp3/Transcription Factor Staining Buffer Sets (eBioscience) according to the manufacturer’s instructions.

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: PLOS ONE

Article Title: Release from persistent T cell receptor engagement and blockade of aryl hydrocarbon receptor activity enhance IL-6-dependent mouse follicular helper T-like cell differentiation in vitro

doi: 10.1371/journal.pone.0287746

Figure Lengend Snippet: ( A ) CH-223191-treated Tfh-like cells were generated as described in the legend of . CH-223191 (5 μM) was added in the first culture. ( B and C ) CD4 + T cells were purified from mesenteric lymph nodes (MLN) and spleens (SPL) of OVA-immunized mice using a EasySep Mouse CD4 + T Cell Enrichment kit. ( D ) Th0 cells were generated in RPMI medium as described in Materials and Methods. Their expression of BCL6 and RORγt protein was determined by flow cytometry, and density plots (A and D) or dot plots (B and C) of their expression are shown. ( E ) BCL6 expression (MFI) levels in Th0, CH-2231-treated Tfh-like cells, and BCL6 + RORγt − CD4 + T cells from MLN and SPL are compared. Data are presented as mean ± SD of three to seven samples. Results shown are representative of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Intracellular staining was performed with anti-BCL6 (clone K112-91) PE and anti-RORγt (Q31-378) Alexa Fluor 647 (both from BD Biosciences) or with anti-IL-21 (clone FFA21) PE (eBioscience) and anti-IL-17A (clone TC11-18H10.1) APC (BioLegend) using Foxp3/Transcription Factor Staining Buffer Sets (eBioscience) according to the manufacturer’s instructions.

Techniques: Generated, Purification, Expressing, Flow Cytometry