apc anti mouse cd4 monoclonal antibody  (Elabscience Biotechnology)

Journal: Immunity

Article Title: Membrane-bound mRNA immunogens lower the threshold to activate HIV Env V2 apex-directed broadly neutralizing B cell precursors in humanized mice

doi: 10.1016/j.immuni.2022.09.003

Figure Lengend Snippet: GT2 immunization activates PCT64 precursor B cells (A) Human PCT64 LMCA IGH (green), murine IGH (dark gray), human PCT64 LMCA IGK (purple) and murine IGK (light gray) sequences amplified from single-cell sorted B220 + naive B cells from two PCT64 LMCA mice. n = pairs amplified. See also Figures S1 and A–S2D. (B) Representative FACS plots of epitope-specific GT2-positive and GT2-KO-negative peripheral B cells in naive PCT64 LMCA or C57BL/6J mice. Events were pre-gated on lymphocytes/singlets/CD4 - CD8 - F4/80 - Gr1 - /B220 + B cells. (C) GT2-specific blood peripheral B cells from PCT64 LMCA and WT C57BL/6J mice (n = 4). Bars are mean ± SD. (D) Human PCT64 LMCA IGH (green), human PCT64 LMCA IGK (purple) and murine IGK (light gray) sequences from single-cell sorted GT2-specific naive B cells in PCT64 LMCA mice (n = 2). n = pairs amplified. (E) Schematic of PCT64 LMCA B cell adoptive transfer and immunization. Experiments performed in triplicate; n = 4. A representative experiment is shown in (F–H). (F) Representative plots of splenic B cells obtained at 8, 16, and 42 dpi with GT2 trimers. Events pre-gated on lymphocytes/singlets/live/CD4 - CD8 - F4/80 - Gr1 - /B220 + B cells and represent GC, CD45.1, and CD45.2 cells in GC, and frequency of GT2 + CD45.2 cells present in GC. For control groups see Figure S2 E. (G) B cell subsets responsive to GT2-immunization at 7, 16, and 42 dpi (n = 4). Left to right: total GCs, CD45.2 + B cells in GCs, and GT2-binding CD45.2 + B cells. Bars are mean ± SD. p values calculated by Mann-Whitney test, ∗∗ p < 0.01. See also Figure S2 E. (H) ELISA quantification of GT2-binding (left) and GT2-KO-binding (right) serum IgG from PCT64 LMCA−HL recipient mice versus WT C57BL/6J mice (n = 4). Line represents mean values. Area under the curve (AUC) was assessed prior to immunization with GT2 trimers and at 7, 14, 21, and 42 dpi.

Article Snippet: Rat monoclonal anti-mouse CD4 APC-eF780 (clone: RM4-5) , Thermo Fisher Scientific , CAT# 47-0042-80; RRID: AB_1272219.

Techniques: Amplification, Adoptive Transfer Assay, Control, Binding Assay, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: Immunity

Article Title: Membrane-bound mRNA immunogens lower the threshold to activate HIV Env V2 apex-directed broadly neutralizing B cell precursors in humanized mice

doi: 10.1016/j.immuni.2022.09.003

Figure Lengend Snippet: PCT64 precursor responses to GT2 are driven by the heavy chain (A) 10x Genomics single-cell BCR sequences from 4703 splenic B cells from a naive PCT64 LMCA−H mouse. Relative bubble size indicates IGHV gene frequency. Human PCT64 IGHV gene frequency in green (82.6%). Murine IGHV genes in various colors. (B) Left: representative fluorescence-activated cell sorting (FACS) plot of GT2-binding and GT2-KO-negative peripheral B cells in naive PCT64 LMCA−H (n = 4). Events were pre-gated on lymphocytes/singlets/CD4 − CD8 - F4/80 - Gr1 - /B220 + B cells. Right: quantification of GT2-specific blood peripheral B cells from PCT64 LMCA−H and C57BL/6J WT mice. Bars are mean ± SD. (C) Paired human PCT64 LMCA IGH (green) and murine IGK (variable) sequences amplified from single-cell sorted GT2-specific naive B cells from PCT64 LMCA−H mice. n = pairs amplified. (D) Murine IGK V genes paired with human PCT64 IGH in a naive PCT64 LMCA−H mouse (n = 3,888). Relative bubble size indicates frequency of IGK V gene usage. Relevant IGK V genes marked in color and analyzed in (E) and (F). See also Figure S4 . (E) Murine IGK V genes paired with human PCT64 IGH isolated from GCs at 8 and 42 dpi. Some frequently enriched IGK V genes are highlighted: V2-109 (red), V12-44 (blue), V14-111 (yellow), V4-74 (light green), and V1-135 (teal). See also Figure S5 A. (F) SPR affinity against GT2 for 23 antibodies with various murine IGKV pairings isolated from naive PCT64 LMCA−H mice at 42 dpi, compared to human PCT64 LMCA (black, square). (G) Frequencies of IGH aa mutations per residue 42 dpi. HCDRs are boxed in gray. Red = key mutations present in mature PCT64. aa positions 31, 35, 52B, and 100D analyzed in (J). See also Figures S5 B–S5E. (H) Distribution of select PCT64 LMCA B cell IGH aa mutations in positions 31, 35, 52B, and 100D at 8 and 42 dpi. Red = present in mature PCT64; blue = present in early PCT64 isolates; black = original LMCA aa; gray = all others.

Article Snippet: Rat monoclonal anti-mouse CD4 APC-eF780 (clone: RM4-5) , Thermo Fisher Scientific , CAT# 47-0042-80; RRID: AB_1272219.

Techniques: Fluorescence, FACS, Binding Assay, Amplification, Isolation, Residue

Journal: Immunity

Article Title: Membrane-bound mRNA immunogens lower the threshold to activate HIV Env V2 apex-directed broadly neutralizing B cell precursors in humanized mice

doi: 10.1016/j.immuni.2022.09.003

Figure Lengend Snippet: GT5-mRNA immunization activates a J-region-reverted PCT64 precursor (A) Sequence alignment of the HCDR3 of PCT64 LMCA.JREV , PCT64 LMCA and germline VH3-15, DH3-3 and JH6. (B) Human PCT64 LMCA.JREV HC (teal), murine HC (dark gray), human PCT64 LMCA.JREV IGK (purple) and murine LC (light gray) sequences amplified from single cell sorted B220 + naive B cells from two PCT64 LMCA.JREV mice. n = pairs amplified. See also Figures S7 A and S7B. (C) SPR affinity measurement against GT5 of LMCA and LMCA.JREV IGH paired with various murine IGK. (D) FACS plots with epitope specific GT2-binding (top) and GT5 binding of peripheral B cells in naive PCT64 LMCA.JREV mouse model. Events were pre-gated on lymphocytes/singlets/CD4 − CD8 - F4/80 - Gr1 - /B220 + B cells. See also Figures S7 C–S7F. (E) Schematic of study design. Recipient mice at 100, 20, or 10 PCT64 LMCA.JREV per 10 6 B cells prior to immunization i.p. with GT5 trimers or IM with GT5 mRNA; responses analyzed 13 dpi. Experiments performed in triplicate with one presented; n = 4 or 5. See also Figures S7 G and S7H. (F) Representative FACS plots of GCs, CD45.2 PCT64 LMCA.JREV present in GC, and GT5 specific responses at 13 dpi after immunization with GT5 protein i.p. (pink) or GT5 mRNA IM (teal). Spleen were analyzed for i.p. responses and inguinal LN for IM responses. (G) Quantification of responses in GCs as in (E) and (F) (n = 4 or 5). Bars are mean ± SD. (H) Quantification of GC responses, frequency of CD45.2 PCT64 LMCA.JREV B cells in GC and GT5 specific responses at 42 dpi (n = 5). Bars are mean ± SD.

Article Snippet: Rat monoclonal anti-mouse CD4 APC-eF780 (clone: RM4-5) , Thermo Fisher Scientific , CAT# 47-0042-80; RRID: AB_1272219.

Techniques: Sequencing, Amplification, Binding Assay

Journal: Immunity

Article Title: Membrane-bound mRNA immunogens lower the threshold to activate HIV Env V2 apex-directed broadly neutralizing B cell precursors in humanized mice

doi: 10.1016/j.immuni.2022.09.003

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Article Snippet: Rat monoclonal anti-mouse CD4 APC-eF780 (clone: RM4-5) , Thermo Fisher Scientific , CAT# 47-0042-80; RRID: AB_1272219.

Techniques: Purification, Recombinant, Produced, Staining, Reverse Transcription, Flow Cytometry, Cell Isolation, Amplification, Software, Adhesive, Spectrophotometry, Electron Microscopy

Journal: eLife

Article Title: Murine alveolar macrophages rapidly accumulate intranasally administered SARS-CoV-2 Spike protein leading to neutrophil recruitment and damage

doi: 10.7554/eLife.86764

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Article Snippet: Antibody , APC Anti-Human CD4 (mouse monoclonal) , BD Bioscience , RRID: AB_398521 , FACS (1:500).

Techniques: Blocking Assay, Imaging, Staining, Recombinant, Plasmid Preparation, Expressing, Luciferase, Cell Culture, Transfection

Journal: Cardiovascular Research

Article Title: Neutrophil inhibition improves acute inflammation in a murine model of viral myocarditis

doi: 10.1093/cvr/cvac052

Figure Lengend Snippet: List of key sources and materials

Article Snippet: Monoclonal rat anti-mouse CD4 APC-H7 , BD Biosciences , Cat# 560181.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant

Journal: eLife

Article Title: Tumor-derived extracellular vesicles regulate tumor-infiltrating regulatory T cells via the inhibitory immunoreceptor CD300a

doi: 10.7554/eLife.61999

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Article Snippet: Antibody , Rat anti-CD4-APC (RM4-5, mouse monoclonal) , BD Bioscience , Cat# 553,051RRID: AB_398528 , FACS (1:5).

Techniques: Purification, Sequencing, Recombinant, Isolation, Software

Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet: ( a ) Expression of CD5 on CXCR4−CCR7 + CD4 CD8+TG6 (96 hr), F5 (72 hr and 96 hr), and OT-1 (72 hr and 96 hr) donor cells developed in thymic slices. Values for each experiment are normalized to the average CD5 expression of the CXCR4−CCR7 + CD4 CD8+polyclonal slice resident (SR) population at the same time points. ( b–d ) Preselection TG6 (white bars, triangle), F5 (gray bars, square), or OT-1 (dark grey bars, circle) thymocytes were overlaid onto selecting or nonselecting thymic slices, harvested at 2, 24, 48, 72, or 96 hr post-thymocyte overlay, and analyzed using flow cytometry. Left three graphs show individual values for each transgenic. Horizontal lines in ( a ) and ( b ) indicate the average value for nonselecting slices. Right line graph shows the average for all three transgenics overlaid. ( b ) Percent of CD69+ cells within the CD4+CD8+ and CD4−CD8+populations. ( c ) Percentage of CXCR4−CCR7 + cells within CD4+CD8+ and CD4−CD8+populations. ( d ) Percentage of CXCR4−CCR7 + CD4 CD8+ cells out of the donor population. Values for each experiment are normalized to the average at the time point with the maximum CD8SP development (72 hr or 96 hr). For ( a ), data are presented as average ± SD and analyzed using an ordinary one-way ANOVA with Tukey’s multiple comparisons (****p<0.000). For ( b–d ), data are presented as average ± SD and analyzed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons (**p<0.01, ***p<0.001, and ****p<0.0001) comparing to 2 hr time point. All data are compiled from three or more experiments. Preselection thymocyte populations were obtained from TG6tg Rag2 −/− H2 b mice, irradiated β2M −/− mice reconstituted with F5tg Rag1 −/− bone marrow, and OT-1tg Rag2 −/− β2M −/− mice. See also and .

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD4 (Clone GK1.5) APC , Tonbo Biosciences , Cat. No. 20–0041 U100 RRID: AB_2621736 , (1:200).

Techniques: Expressing, Flow Cytometry, Transgenic Assay, Irradiation

Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet: ( a ) Representative flow plots illustrating the gating strategy for thymic slice experiments. ( b ) Representative CD4 vs CD8 flow plots of TG6, F5, and OT-1 live donor thymocytes (bolded red gate in a ). ( c ) CD4 vs CD8 representative flow plots for TG6, F5, and OT-1 live donor CXCR4-CCR7 + thymocytes (bolded blue gate in a ). Bolded numbers within plots indicate the percentage of cells within a given gate.

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD4 (Clone GK1.5) APC , Tonbo Biosciences , Cat. No. 20–0041 U100 RRID: AB_2621736 , (1:200).

Techniques:

Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet: Thymuses were harvested from TG6 (triangle, white bar), F5 (square, gray bar), or OT-1 (circle, black bar) transgenic neonatal mice at 7, 14, and 21 days post-birth and from adult mice (6–9 weeks old), and analyzed using flow cytometry. ( a ) Percentage of CD4−CD8+ cells. ( b ) Total number of CD4−CD8+ thymocytes at the indicated time point post-birth, relative to adult, for each transgenic. ( c ) Percentage of CXCR4−CCR7+ cells within CD4+CD8+ and CD4−CD8+ populations. Data are presented as average ± SD and analyzed using an ordinary one-way ANOVA, Dunnett’s multiple comparisons (*p<0.05, **p<0.01, and ****p<0.0001) comparing to adult. All data are compiled from three or more experiments.

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD4 (Clone GK1.5) APC , Tonbo Biosciences , Cat. No. 20–0041 U100 RRID: AB_2621736 , (1:200).

Techniques: Transgenic Assay, Flow Cytometry

Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet: Total number of CD4−CD8+ splenocytes relative to adult for each transgenic. Data compiled from three or more experiments.

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD4 (Clone GK1.5) APC , Tonbo Biosciences , Cat. No. 20–0041 U100 RRID: AB_2621736 , (1:200).

Techniques: Transgenic Assay

Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet: TG6, F5, or OT-1 ( a–c ) or B6 ( d–f ) mice were injected with two doses of 1 mg of EdU intraperitoneally (i.p.) at 0 and 4 hr post-injection (p.i.). ( a ) TCRtg experimental schematic. ( b ) EdU incorporation into TCRtg thymuses. ( c ) (Left three graphs) Percent CD8SP and DP thymocytes out of gated EdU+ cells at various days p.i. in TG6, F5, and OT-1 tg thymuses. (Right) The percent of EdU+CD8 SP relative to max within each experiment. Data are presented as the average within each transgenic and analyzed using an ordinary one-way ANOVA at each time point (*p<0.05, **p<0.01). Tukey’s multiple comparisons showed significant differences between OT-1 and TG6 at day 4 (**), day 5 (*), and day 6 (*); OT-1 and F5 at day 4 (*); and a difference between F5 vs TG6 at Day 6 (p=0.069). ( d ) B6 experimental schematic. ( e ) Representative gating strategy and histograms showing B6 day 10 p.i. CD5 expression on EdU+ and EdU− mature SP cells. ( f ) CD5 expression of EdU+TCRβ+CD8 SP and EdU+CD4 SP thymocytes at 4, 7, and 10 days p.i. CD5 expression shown relative to total TCRb+CD8 SP or CD4SP CD5 expression. In ( f ), data are presented as average ± SD and analyzed using an ordinary one-way ANOVA, Tukey’s multiple comparisons (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). All data are compiled from three or more experiments.

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD4 (Clone GK1.5) APC , Tonbo Biosciences , Cat. No. 20–0041 U100 RRID: AB_2621736 , (1:200).

Techniques: Injection, Transgenic Assay, Expressing

Journal: eLife

Article Title: T cell self-reactivity during thymic development dictates the timing of positive selection

doi: 10.7554/eLife.65435

Figure Lengend Snippet:

Article Snippet: Antibody , Rat Monoclonal anti-Mouse CD4 (Clone GK1.5) APC , Tonbo Biosciences , Cat. No. 20–0041 U100 RRID: AB_2621736 , (1:200).

Techniques: Generated, Staining, Flow Cytometry, Software