tonbo cytek pe cy7 cd25 pc61 5 Search Results


abs cd25 (percp-cy5.5-pc61.5  (Cytek Biosciences)
90
Cytek Biosciences abs cd25 (percp-cy5.5-pc61.5
Abs Cd25 (Percp Cy5.5 Pc61.5, supplied by Cytek Biosciences, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti cd25 pe  (Cytek Biosciences)
94
Cytek Biosciences anti cd25 pe
Analysis of critical transcription factors in wild-type (WT) and Mb1-cre ABCB7 cKO Fr. C cells (B220 + CD19 + CD43 + BP-1 + ). ( A–G ) Intracellular flow cytometry analysis of EBF1 ( A ), E47 (E2A) ( B ), FOXO1 ( C ), PAX5 ( D ), IKAROS ( E ), AIOLOS ( F ), and IRF4 ( G ) expression. Quantification of MdFI is shown on the right of each plot. Isotype controls are shown in gray. Offset histograms are representative of at least three independent experiments (total of 6–10 mice/group). ( H, I ) Flow cytometry analysis of CD2 ( H ) and <t>CD25</t> ( I ) expression. Indicated values are the proportion of Fr. C cells positive for either marker, and quantifications are shown on the right of each plot. Offset histograms are representative of three independent experiments (total of five mice/group). ( J ) Intracellular flow cytometry analysis of TdT expression in Fr. B and Fr. C cells. Indicated values are the proportion of cells positive for TdT expression, and quantifications are shown on the right. Offset histograms are representative of three independent experiments (total of 5–7 mice/group). ( K ) Quantitative real-time PCR analysis of Rag1 and Rag2 expression in sorted Fr. B and Fr. C cells. 18S rRNA was used as an endogenous control, and relative expression values were normalized to expression in WT Fr. B cells. Results were obtained from three independent experiments (total of 3–4 mice/group). ( A–K ) Error bars represent SEM, and p-values are indicated above the data. Statistics were obtained by using an unpaired Student’s t -test.
Anti Cd25 Pe, supplied by Cytek Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pe cd25  (Cytek Biosciences)
90
Cytek Biosciences pe cd25
Analysis of critical transcription factors in wild-type (WT) and Mb1-cre ABCB7 cKO Fr. C cells (B220 + CD19 + CD43 + BP-1 + ). ( A–G ) Intracellular flow cytometry analysis of EBF1 ( A ), E47 (E2A) ( B ), FOXO1 ( C ), PAX5 ( D ), IKAROS ( E ), AIOLOS ( F ), and IRF4 ( G ) expression. Quantification of MdFI is shown on the right of each plot. Isotype controls are shown in gray. Offset histograms are representative of at least three independent experiments (total of 6–10 mice/group). ( H, I ) Flow cytometry analysis of CD2 ( H ) and <t>CD25</t> ( I ) expression. Indicated values are the proportion of Fr. C cells positive for either marker, and quantifications are shown on the right of each plot. Offset histograms are representative of three independent experiments (total of five mice/group). ( J ) Intracellular flow cytometry analysis of TdT expression in Fr. B and Fr. C cells. Indicated values are the proportion of cells positive for TdT expression, and quantifications are shown on the right. Offset histograms are representative of three independent experiments (total of 5–7 mice/group). ( K ) Quantitative real-time PCR analysis of Rag1 and Rag2 expression in sorted Fr. B and Fr. C cells. 18S rRNA was used as an endogenous control, and relative expression values were normalized to expression in WT Fr. B cells. Results were obtained from three independent experiments (total of 3–4 mice/group). ( A–K ) Error bars represent SEM, and p-values are indicated above the data. Statistics were obtained by using an unpaired Student’s t -test.
Pe Cd25, supplied by Cytek Biosciences, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cd25 pc61 5 biotin  (Cytek Biosciences)
93
Cytek Biosciences cd25 pc61 5 biotin
(A) Subcutaneous inoculation (s.c.) with 10 5 p.f.u. of VACV was administered to both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells, as well as <t>CD25</t> + Foxp3 + Treg cells, was conducted in popliteal lymph nodes (pLN) at 6 and 14 days post-infection. The percentage of Th1 cells, CD8 CTL T cells, and Treg cells was assessed. The data represent means ± SEM from at least 8 mice per group, obtained from 3 independently conducted experiments, and were analyzed using unpaired Student’s t-test between genotypes at each time point. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant). (B) Intraperitoneal inoculation (i.p.) with 10 6 p.f.u. of VACV was performed on both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells was conducted in mesenteric lymph nodes (mLN) 6 days post-infection. The percentage of Th1 and CD8 CTL cells was determined. Data represent means ± SEM of at least 6 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is indicated by asterisks (*P < 0.05; **P < 0.01). (C) Infection with 10 5 p.f.u. of VACV through skin scarification (s.s.) in the tail was performed on WT and LysM-Lmna -/- mice. Flow cytometry analysis of the percentages of IFNγ-producing CD4 and CD8 T cells was conducted in extracted inguinal lymph nodes (iLN) at 6 and 14 days post-infection. (D) Viral load measurement in the tail skin was performed at 6 and 14 days post-infection. The data represent means ± SEM of at least 4 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant).
Cd25 Pc61 5 Biotin, supplied by Cytek Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti cd25 pc61 5  (Cytek Biosciences)
90
Cytek Biosciences anti cd25 pc61 5
a,b) . Flow cytometry quantification validating that M-II KO mice have decreased MHC-II expression in tumour-infiltrating immune cells (CD45 + ), including macrophages (Mϕ, CD45 + CD11b + Ly6G F4/80 + ), dendritic cells (DC, CD45 + CD11b + Ly6G − F4/80 − CD11c + ), and B cells (CD45 + B220 + ) (CTRL: n=10; M-II KO : n=11). c) . Flow cytometry gating of suspension cells dissociated from thymus, characterized as CD45 + EpCAM − immune cells and CD45 − EpCAM + epithelial cells. d,e) . Quantification of MHC-II expression of thymus showing that MHC-II expression is inhibited in immune cells but preserved in epithelial cells in M-II KO mice (CTRL: n=7; M-II KO : n=6). f) . Quantification of different types of tumour-infiltrating stroma cells (n=10/group). g) . Quantification of MHC-II expression in different cell types (CTRL: n=10; Tie2Cre;H2Ab +/floxP : n=5; M-II KO : n=11). h) . Quantification of T cells in spleens from 5 – 6 week-old female mice showing the number of T cells is independent of MHC-II expression on Tie2Cre + cells (CTRL: n=7; M-II KO : n=6). i) . Quantification of activated CD4 + -TLs and effector CD4 + -TLs from tumours of similar sizes. (activated CD4 + -TL: CD45 + CD3 + CD4 + <t>CD25</t> + FoxP3 − Treg: CD45 + CD3 + CD4 + CD25 + FoxP3 + ; Effector memory cell: CD44 + CD62L − Naïve CD4 + -TL: CD44 − CD62L + ) (n=11/group). j) . The percentages of CD4 + -TL activation markers in spleen showing a similar pattern as in tumour (i) (CTRL: n=7; M-II KO : n=6). k) . Quantification of different E0771 tumour-infiltrating T helper cells (IFNγ + Th1, IL4 + Th2 and IL17A + Th17) (n=11/group). l) . Quantification of E0771 tumour-infiltrating CD4 + -TL cells, macrophage, dendritic cells, B cells and neutrophils (CD45 + CD11b + Ly6G high ) (n=11/group). Data are presented as means ± s.e.m. The genetic backgrounds of mice are denoted with different colors shown on the right of (l) . WT, Tie2Cre and H2Ab floxP/floxP were combined as CTRL group. P values were calculated using two-tailed unpaired Student’s t-test (a–b, d–f, h–k) or two-tailed unpaired Mann–Whitney U test ( l ). n.s., not significant.
Anti Cd25 Pc61 5, supplied by Cytek Biosciences, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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violetfluor 450 coupled anti cd25 pc61 5  (Cytek Biosciences)
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Cytek Biosciences violetfluor 450 coupled anti cd25 pc61 5
a,b) . Flow cytometry quantification validating that M-II KO mice have decreased MHC-II expression in tumour-infiltrating immune cells (CD45 + ), including macrophages (Mϕ, CD45 + CD11b + Ly6G F4/80 + ), dendritic cells (DC, CD45 + CD11b + Ly6G − F4/80 − CD11c + ), and B cells (CD45 + B220 + ) (CTRL: n=10; M-II KO : n=11). c) . Flow cytometry gating of suspension cells dissociated from thymus, characterized as CD45 + EpCAM − immune cells and CD45 − EpCAM + epithelial cells. d,e) . Quantification of MHC-II expression of thymus showing that MHC-II expression is inhibited in immune cells but preserved in epithelial cells in M-II KO mice (CTRL: n=7; M-II KO : n=6). f) . Quantification of different types of tumour-infiltrating stroma cells (n=10/group). g) . Quantification of MHC-II expression in different cell types (CTRL: n=10; Tie2Cre;H2Ab +/floxP : n=5; M-II KO : n=11). h) . Quantification of T cells in spleens from 5 – 6 week-old female mice showing the number of T cells is independent of MHC-II expression on Tie2Cre + cells (CTRL: n=7; M-II KO : n=6). i) . Quantification of activated CD4 + -TLs and effector CD4 + -TLs from tumours of similar sizes. (activated CD4 + -TL: CD45 + CD3 + CD4 + <t>CD25</t> + FoxP3 − Treg: CD45 + CD3 + CD4 + CD25 + FoxP3 + ; Effector memory cell: CD44 + CD62L − Naïve CD4 + -TL: CD44 − CD62L + ) (n=11/group). j) . The percentages of CD4 + -TL activation markers in spleen showing a similar pattern as in tumour (i) (CTRL: n=7; M-II KO : n=6). k) . Quantification of different E0771 tumour-infiltrating T helper cells (IFNγ + Th1, IL4 + Th2 and IL17A + Th17) (n=11/group). l) . Quantification of E0771 tumour-infiltrating CD4 + -TL cells, macrophage, dendritic cells, B cells and neutrophils (CD45 + CD11b + Ly6G high ) (n=11/group). Data are presented as means ± s.e.m. The genetic backgrounds of mice are denoted with different colors shown on the right of (l) . WT, Tie2Cre and H2Ab floxP/floxP were combined as CTRL group. P values were calculated using two-tailed unpaired Student’s t-test (a–b, d–f, h–k) or two-tailed unpaired Mann–Whitney U test ( l ). n.s., not significant.
Violetfluor 450 Coupled Anti Cd25 Pc61 5, supplied by Cytek Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cd25 pc61 5 apc  (Cytek Biosciences)
94
Cytek Biosciences cd25 pc61 5 apc
(A) Subcutaneous inoculation (s.c.) with 10 5 p.f.u. of VACV was administered to both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells, as well as <t>CD25</t> + Foxp3 + Treg cells, was conducted in popliteal lymph nodes (pLN) at 6 and 14 days post-infection. The percentage of Th1 cells, CD8 CTL T cells, and Treg cells was assessed. The data represent means ± SEM from at least 8 mice per group, obtained from 3 independently conducted experiments, and were analyzed using unpaired Student’s t-test between genotypes at each time point. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant). (B) Intraperitoneal inoculation (i.p.) with 10 6 p.f.u. of VACV was performed on both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells was conducted in mesenteric lymph nodes (mLN) 6 days post-infection. The percentage of Th1 and CD8 CTL cells was determined. Data represent means ± SEM of at least 6 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is indicated by asterisks (*P < 0.05; **P < 0.01). (C) Infection with 10 5 p.f.u. of VACV through skin scarification (s.s.) in the tail was performed on WT and LysM-Lmna -/- mice. Flow cytometry analysis of the percentages of IFNγ-producing CD4 and CD8 T cells was conducted in extracted inguinal lymph nodes (iLN) at 6 and 14 days post-infection. (D) Viral load measurement in the tail skin was performed at 6 and 14 days post-infection. The data represent means ± SEM of at least 4 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant).
Cd25 Pc61 5 Apc, supplied by Cytek Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti cd25 pc61 5 percp c5 5 tonbo biosciences  (Cytek Biosciences)
93
Cytek Biosciences anti cd25 pc61 5 percp c5 5 tonbo biosciences
(A) Subcutaneous inoculation (s.c.) with 10 5 p.f.u. of VACV was administered to both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells, as well as <t>CD25</t> + Foxp3 + Treg cells, was conducted in popliteal lymph nodes (pLN) at 6 and 14 days post-infection. The percentage of Th1 cells, CD8 CTL T cells, and Treg cells was assessed. The data represent means ± SEM from at least 8 mice per group, obtained from 3 independently conducted experiments, and were analyzed using unpaired Student’s t-test between genotypes at each time point. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant). (B) Intraperitoneal inoculation (i.p.) with 10 6 p.f.u. of VACV was performed on both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells was conducted in mesenteric lymph nodes (mLN) 6 days post-infection. The percentage of Th1 and CD8 CTL cells was determined. Data represent means ± SEM of at least 6 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is indicated by asterisks (*P < 0.05; **P < 0.01). (C) Infection with 10 5 p.f.u. of VACV through skin scarification (s.s.) in the tail was performed on WT and LysM-Lmna -/- mice. Flow cytometry analysis of the percentages of IFNγ-producing CD4 and CD8 T cells was conducted in extracted inguinal lymph nodes (iLN) at 6 and 14 days post-infection. (D) Viral load measurement in the tail skin was performed at 6 and 14 days post-infection. The data represent means ± SEM of at least 4 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant).
Anti Cd25 Pc61 5 Percp C5 5 Tonbo Biosciences, supplied by Cytek Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cd49d r1-2 antibody  (Thermo Fisher)
90
Thermo Fisher cd49d r1-2 antibody
(A) Subcutaneous inoculation (s.c.) with 10 5 p.f.u. of VACV was administered to both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells, as well as <t>CD25</t> + Foxp3 + Treg cells, was conducted in popliteal lymph nodes (pLN) at 6 and 14 days post-infection. The percentage of Th1 cells, CD8 CTL T cells, and Treg cells was assessed. The data represent means ± SEM from at least 8 mice per group, obtained from 3 independently conducted experiments, and were analyzed using unpaired Student’s t-test between genotypes at each time point. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant). (B) Intraperitoneal inoculation (i.p.) with 10 6 p.f.u. of VACV was performed on both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells was conducted in mesenteric lymph nodes (mLN) 6 days post-infection. The percentage of Th1 and CD8 CTL cells was determined. Data represent means ± SEM of at least 6 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is indicated by asterisks (*P < 0.05; **P < 0.01). (C) Infection with 10 5 p.f.u. of VACV through skin scarification (s.s.) in the tail was performed on WT and LysM-Lmna -/- mice. Flow cytometry analysis of the percentages of IFNγ-producing CD4 and CD8 T cells was conducted in extracted inguinal lymph nodes (iLN) at 6 and 14 days post-infection. (D) Viral load measurement in the tail skin was performed at 6 and 14 days post-infection. The data represent means ± SEM of at least 4 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant).
Cd49d R1 2 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cd25  (Cytek Biosciences)
94
Cytek Biosciences cd25
(A) Subcutaneous inoculation (s.c.) with 10 5 p.f.u. of VACV was administered to both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells, as well as <t>CD25</t> + Foxp3 + Treg cells, was conducted in popliteal lymph nodes (pLN) at 6 and 14 days post-infection. The percentage of Th1 cells, CD8 CTL T cells, and Treg cells was assessed. The data represent means ± SEM from at least 8 mice per group, obtained from 3 independently conducted experiments, and were analyzed using unpaired Student’s t-test between genotypes at each time point. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant). (B) Intraperitoneal inoculation (i.p.) with 10 6 p.f.u. of VACV was performed on both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells was conducted in mesenteric lymph nodes (mLN) 6 days post-infection. The percentage of Th1 and CD8 CTL cells was determined. Data represent means ± SEM of at least 6 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is indicated by asterisks (*P < 0.05; **P < 0.01). (C) Infection with 10 5 p.f.u. of VACV through skin scarification (s.s.) in the tail was performed on WT and LysM-Lmna -/- mice. Flow cytometry analysis of the percentages of IFNγ-producing CD4 and CD8 T cells was conducted in extracted inguinal lymph nodes (iLN) at 6 and 14 days post-infection. (D) Viral load measurement in the tail skin was performed at 6 and 14 days post-infection. The data represent means ± SEM of at least 4 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant).
Cd25, supplied by Cytek Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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f4/80-percp-cyanine5.5 bm8 antibody  (Thermo Fisher)
90
Thermo Fisher f4/80-percp-cyanine5.5 bm8 antibody
(A) 2C CD8 T cell proliferation to BALB/c stimulation was measured by CFSE dilution in MLRs set up as described in Methods. In some cases, MACS sorted splenic M-or G-MDSCs from B6 mice were added to the MLRs to determine their suppression of 2C T cell proliferation. In additional cases, rIFN-α (1000 U/ml) or anti-IFNaR1 (20 mg/ml) was further added to the cultures to determine their effects on the MDSC-mediated suppression of 2C proliferation. Representative histograms were gated on 2C CD8+ T cells. Percentage of proliferation in the right bar graph was calculated as (2C CD8+ T cells with diluted CFSE)/(total 2C CD8+ T cells). (B) B6 <t>CD4+CD25+Foxp3+</t> Treg proliferation to BALB/c stimulation was measured by CFSE dilution in MLRs set up as described in Methods. In some cases, MACS sorted splenic M-or G-MDSCs from B6 mice were added to the MLRs to determine their effect on CD4+CD25+Foxp3+ Treg proliferation. In additional cases, rIFN-α or anti-IFNaR1 was further added to the cultures to determine their effects on the MDSC-mediated promotion of CD4+CD25+Foxp3+ Treg proliferation. Representative histograms were gated on CD4+CD25+Foxp3+ Tregs. Percentage of proliferation in the right bar graph was calculated as (CD4+CD25+Foxp3+ Tregs with diluted CFSE)/(total CD4+CD25+Foxp3+ Tregs). Data for (A-B) were obtained and averaged from three independent experiments. Statistical significance was determined by either two-way ANOVA with Bonferroni post-test or one-way ANOVA with Bonferroni post-test. *P ≤ 0.05, ***P ≤ 0.001.
F4/80 Percp Cyanine5.5 Bm8 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Analysis of critical transcription factors in wild-type (WT) and Mb1-cre ABCB7 cKO Fr. C cells (B220 + CD19 + CD43 + BP-1 + ). ( A–G ) Intracellular flow cytometry analysis of EBF1 ( A ), E47 (E2A) ( B ), FOXO1 ( C ), PAX5 ( D ), IKAROS ( E ), AIOLOS ( F ), and IRF4 ( G ) expression. Quantification of MdFI is shown on the right of each plot. Isotype controls are shown in gray. Offset histograms are representative of at least three independent experiments (total of 6–10 mice/group). ( H, I ) Flow cytometry analysis of CD2 ( H ) and CD25 ( I ) expression. Indicated values are the proportion of Fr. C cells positive for either marker, and quantifications are shown on the right of each plot. Offset histograms are representative of three independent experiments (total of five mice/group). ( J ) Intracellular flow cytometry analysis of TdT expression in Fr. B and Fr. C cells. Indicated values are the proportion of cells positive for TdT expression, and quantifications are shown on the right. Offset histograms are representative of three independent experiments (total of 5–7 mice/group). ( K ) Quantitative real-time PCR analysis of Rag1 and Rag2 expression in sorted Fr. B and Fr. C cells. 18S rRNA was used as an endogenous control, and relative expression values were normalized to expression in WT Fr. B cells. Results were obtained from three independent experiments (total of 3–4 mice/group). ( A–K ) Error bars represent SEM, and p-values are indicated above the data. Statistics were obtained by using an unpaired Student’s t -test.

Journal: eLife

Article Title: The mitochondrial iron transporter ABCB7 is required for B cell development, proliferation, and class switch recombination in mice

doi: 10.7554/eLife.69621

Figure Lengend Snippet: Analysis of critical transcription factors in wild-type (WT) and Mb1-cre ABCB7 cKO Fr. C cells (B220 + CD19 + CD43 + BP-1 + ). ( A–G ) Intracellular flow cytometry analysis of EBF1 ( A ), E47 (E2A) ( B ), FOXO1 ( C ), PAX5 ( D ), IKAROS ( E ), AIOLOS ( F ), and IRF4 ( G ) expression. Quantification of MdFI is shown on the right of each plot. Isotype controls are shown in gray. Offset histograms are representative of at least three independent experiments (total of 6–10 mice/group). ( H, I ) Flow cytometry analysis of CD2 ( H ) and CD25 ( I ) expression. Indicated values are the proportion of Fr. C cells positive for either marker, and quantifications are shown on the right of each plot. Offset histograms are representative of three independent experiments (total of five mice/group). ( J ) Intracellular flow cytometry analysis of TdT expression in Fr. B and Fr. C cells. Indicated values are the proportion of cells positive for TdT expression, and quantifications are shown on the right. Offset histograms are representative of three independent experiments (total of 5–7 mice/group). ( K ) Quantitative real-time PCR analysis of Rag1 and Rag2 expression in sorted Fr. B and Fr. C cells. 18S rRNA was used as an endogenous control, and relative expression values were normalized to expression in WT Fr. B cells. Results were obtained from three independent experiments (total of 3–4 mice/group). ( A–K ) Error bars represent SEM, and p-values are indicated above the data. Statistics were obtained by using an unpaired Student’s t -test.

Article Snippet: Antibody , Anti-CD25 PE (Rat monoclonal, PC61.5) , Tonbo Biosciences , Cat#:50-0251; RRID: AB_2621757 , FC (1:200).

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

Journal: eLife

Article Title: The mitochondrial iron transporter ABCB7 is required for B cell development, proliferation, and class switch recombination in mice

doi: 10.7554/eLife.69621

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-CD25 PE (Rat monoclonal, PC61.5) , Tonbo Biosciences , Cat#:50-0251; RRID: AB_2621757 , FC (1:200).

Techniques: Transgenic Assay, Selection, Concentration Assay, Sequencing, Recombinant, Flow Cytometry, EdU Assay, DNA Purification, Isolation, Colony-forming Unit Assay, Cell Cycle Assay, Software

(A) Subcutaneous inoculation (s.c.) with 10 5 p.f.u. of VACV was administered to both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells, as well as CD25 + Foxp3 + Treg cells, was conducted in popliteal lymph nodes (pLN) at 6 and 14 days post-infection. The percentage of Th1 cells, CD8 CTL T cells, and Treg cells was assessed. The data represent means ± SEM from at least 8 mice per group, obtained from 3 independently conducted experiments, and were analyzed using unpaired Student’s t-test between genotypes at each time point. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant). (B) Intraperitoneal inoculation (i.p.) with 10 6 p.f.u. of VACV was performed on both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells was conducted in mesenteric lymph nodes (mLN) 6 days post-infection. The percentage of Th1 and CD8 CTL cells was determined. Data represent means ± SEM of at least 6 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is indicated by asterisks (*P < 0.05; **P < 0.01). (C) Infection with 10 5 p.f.u. of VACV through skin scarification (s.s.) in the tail was performed on WT and LysM-Lmna -/- mice. Flow cytometry analysis of the percentages of IFNγ-producing CD4 and CD8 T cells was conducted in extracted inguinal lymph nodes (iLN) at 6 and 14 days post-infection. (D) Viral load measurement in the tail skin was performed at 6 and 14 days post-infection. The data represent means ± SEM of at least 4 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant).

Journal: bioRxiv

Article Title: Role of Lamin A/C on dendritic cell function in antiviral immunity

doi: 10.1101/2024.05.14.593747

Figure Lengend Snippet: (A) Subcutaneous inoculation (s.c.) with 10 5 p.f.u. of VACV was administered to both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells, as well as CD25 + Foxp3 + Treg cells, was conducted in popliteal lymph nodes (pLN) at 6 and 14 days post-infection. The percentage of Th1 cells, CD8 CTL T cells, and Treg cells was assessed. The data represent means ± SEM from at least 8 mice per group, obtained from 3 independently conducted experiments, and were analyzed using unpaired Student’s t-test between genotypes at each time point. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant). (B) Intraperitoneal inoculation (i.p.) with 10 6 p.f.u. of VACV was performed on both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells was conducted in mesenteric lymph nodes (mLN) 6 days post-infection. The percentage of Th1 and CD8 CTL cells was determined. Data represent means ± SEM of at least 6 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is indicated by asterisks (*P < 0.05; **P < 0.01). (C) Infection with 10 5 p.f.u. of VACV through skin scarification (s.s.) in the tail was performed on WT and LysM-Lmna -/- mice. Flow cytometry analysis of the percentages of IFNγ-producing CD4 and CD8 T cells was conducted in extracted inguinal lymph nodes (iLN) at 6 and 14 days post-infection. (D) Viral load measurement in the tail skin was performed at 6 and 14 days post-infection. The data represent means ± SEM of at least 4 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant).

Article Snippet: Antibodies against: CD4 (Clone RM4-5, GK1.5) -v450, -APC and PE; CD25 (PC61.5) -APC; CD69 (H1.2F3)-FITC; CD28 (37.51); CD3 (145-2C11); CD45.1 (A20)-Pe-Cy7, -v450, -FITC; CD45.2 (104) -v450, -redFluor710; CD11b (M1/70)-v450; CD86 GL-1 PE; IFNγ XMG1.2 FITC, -APC; Foxp3 (3G3)-FITC; MHC-II (I-A/I-E, M5/115.15.2)-APC; GR1 (RB6-8C5)-Biotin; CD80 (16-10A1)-Biotin; B220 (RA3-6B2)-Biotin; F4/80 (BM8.1)-Biotin; CD11b (M1/70)-Biotin; CD19 (1D3)-Biotin; CD25 (PC61.5)-Biotin from Tonbo.

Techniques: Flow Cytometry, Infection, Derivative Assay

Flow cytometry was employed to assess the percentage of activated CD4 T cells, specifically gated on CD25 + or CD69 + cells; Th1-differentiated cells, specifically gated on IFNγ+ cells; and proliferating CellTrace Violet + T cells. Maturation of GM-CSF-derived WT- and LysM-Lmna -/- -BMDCs was undertaken with (A) sonicated VACV extract, (B) Pam3CSK4, or (C) lipopolysaccharide (LPS), and, in all conditions, pulsed with OVA323–339 cognate OT-IIp. (D) WT- or Lmna -/- -BMDCs, matured with LPS and pulsed with the OVA 323–339 cognate OT-II peptide, were s.c. inoculated into CD45.1 OT-II recipient mice. Six days later, pLNs were extracted, and IFNγ-producing CD4 T cells were analyzed by flow cytometry. The data represent means ± SEM and are presented from a representative experiment out of 3 (A and B, n=3) or out of 4 (C, n=4) or out of 2 (D, n=4). Activation data were analyzed using One Way ANOVA and Bonferroni post-test, and proliferation and Th1 differentiation were assessed by unpaired Student’s t-test. Statistical significance was denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant).

Journal: bioRxiv

Article Title: Role of Lamin A/C on dendritic cell function in antiviral immunity

doi: 10.1101/2024.05.14.593747

Figure Lengend Snippet: Flow cytometry was employed to assess the percentage of activated CD4 T cells, specifically gated on CD25 + or CD69 + cells; Th1-differentiated cells, specifically gated on IFNγ+ cells; and proliferating CellTrace Violet + T cells. Maturation of GM-CSF-derived WT- and LysM-Lmna -/- -BMDCs was undertaken with (A) sonicated VACV extract, (B) Pam3CSK4, or (C) lipopolysaccharide (LPS), and, in all conditions, pulsed with OVA323–339 cognate OT-IIp. (D) WT- or Lmna -/- -BMDCs, matured with LPS and pulsed with the OVA 323–339 cognate OT-II peptide, were s.c. inoculated into CD45.1 OT-II recipient mice. Six days later, pLNs were extracted, and IFNγ-producing CD4 T cells were analyzed by flow cytometry. The data represent means ± SEM and are presented from a representative experiment out of 3 (A and B, n=3) or out of 4 (C, n=4) or out of 2 (D, n=4). Activation data were analyzed using One Way ANOVA and Bonferroni post-test, and proliferation and Th1 differentiation were assessed by unpaired Student’s t-test. Statistical significance was denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant).

Article Snippet: Antibodies against: CD4 (Clone RM4-5, GK1.5) -v450, -APC and PE; CD25 (PC61.5) -APC; CD69 (H1.2F3)-FITC; CD28 (37.51); CD3 (145-2C11); CD45.1 (A20)-Pe-Cy7, -v450, -FITC; CD45.2 (104) -v450, -redFluor710; CD11b (M1/70)-v450; CD86 GL-1 PE; IFNγ XMG1.2 FITC, -APC; Foxp3 (3G3)-FITC; MHC-II (I-A/I-E, M5/115.15.2)-APC; GR1 (RB6-8C5)-Biotin; CD80 (16-10A1)-Biotin; B220 (RA3-6B2)-Biotin; F4/80 (BM8.1)-Biotin; CD11b (M1/70)-Biotin; CD19 (1D3)-Biotin; CD25 (PC61.5)-Biotin from Tonbo.

Techniques: Flow Cytometry, Derivative Assay, Sonication, Activation Assay

a,b) . Flow cytometry quantification validating that M-II KO mice have decreased MHC-II expression in tumour-infiltrating immune cells (CD45 + ), including macrophages (Mϕ, CD45 + CD11b + Ly6G F4/80 + ), dendritic cells (DC, CD45 + CD11b + Ly6G − F4/80 − CD11c + ), and B cells (CD45 + B220 + ) (CTRL: n=10; M-II KO : n=11). c) . Flow cytometry gating of suspension cells dissociated from thymus, characterized as CD45 + EpCAM − immune cells and CD45 − EpCAM + epithelial cells. d,e) . Quantification of MHC-II expression of thymus showing that MHC-II expression is inhibited in immune cells but preserved in epithelial cells in M-II KO mice (CTRL: n=7; M-II KO : n=6). f) . Quantification of different types of tumour-infiltrating stroma cells (n=10/group). g) . Quantification of MHC-II expression in different cell types (CTRL: n=10; Tie2Cre;H2Ab +/floxP : n=5; M-II KO : n=11). h) . Quantification of T cells in spleens from 5 – 6 week-old female mice showing the number of T cells is independent of MHC-II expression on Tie2Cre + cells (CTRL: n=7; M-II KO : n=6). i) . Quantification of activated CD4 + -TLs and effector CD4 + -TLs from tumours of similar sizes. (activated CD4 + -TL: CD45 + CD3 + CD4 + CD25 + FoxP3 − Treg: CD45 + CD3 + CD4 + CD25 + FoxP3 + ; Effector memory cell: CD44 + CD62L − Naïve CD4 + -TL: CD44 − CD62L + ) (n=11/group). j) . The percentages of CD4 + -TL activation markers in spleen showing a similar pattern as in tumour (i) (CTRL: n=7; M-II KO : n=6). k) . Quantification of different E0771 tumour-infiltrating T helper cells (IFNγ + Th1, IL4 + Th2 and IL17A + Th17) (n=11/group). l) . Quantification of E0771 tumour-infiltrating CD4 + -TL cells, macrophage, dendritic cells, B cells and neutrophils (CD45 + CD11b + Ly6G high ) (n=11/group). Data are presented as means ± s.e.m. The genetic backgrounds of mice are denoted with different colors shown on the right of (l) . WT, Tie2Cre and H2Ab floxP/floxP were combined as CTRL group. P values were calculated using two-tailed unpaired Student’s t-test (a–b, d–f, h–k) or two-tailed unpaired Mann–Whitney U test ( l ). n.s., not significant.

Journal: Nature

Article Title: Mutual Regulation of Tumour Vessel Normalization and Immunostimulatory Reprogramming

doi: 10.1038/nature21724

Figure Lengend Snippet: a,b) . Flow cytometry quantification validating that M-II KO mice have decreased MHC-II expression in tumour-infiltrating immune cells (CD45 + ), including macrophages (Mϕ, CD45 + CD11b + Ly6G F4/80 + ), dendritic cells (DC, CD45 + CD11b + Ly6G − F4/80 − CD11c + ), and B cells (CD45 + B220 + ) (CTRL: n=10; M-II KO : n=11). c) . Flow cytometry gating of suspension cells dissociated from thymus, characterized as CD45 + EpCAM − immune cells and CD45 − EpCAM + epithelial cells. d,e) . Quantification of MHC-II expression of thymus showing that MHC-II expression is inhibited in immune cells but preserved in epithelial cells in M-II KO mice (CTRL: n=7; M-II KO : n=6). f) . Quantification of different types of tumour-infiltrating stroma cells (n=10/group). g) . Quantification of MHC-II expression in different cell types (CTRL: n=10; Tie2Cre;H2Ab +/floxP : n=5; M-II KO : n=11). h) . Quantification of T cells in spleens from 5 – 6 week-old female mice showing the number of T cells is independent of MHC-II expression on Tie2Cre + cells (CTRL: n=7; M-II KO : n=6). i) . Quantification of activated CD4 + -TLs and effector CD4 + -TLs from tumours of similar sizes. (activated CD4 + -TL: CD45 + CD3 + CD4 + CD25 + FoxP3 − Treg: CD45 + CD3 + CD4 + CD25 + FoxP3 + ; Effector memory cell: CD44 + CD62L − Naïve CD4 + -TL: CD44 − CD62L + ) (n=11/group). j) . The percentages of CD4 + -TL activation markers in spleen showing a similar pattern as in tumour (i) (CTRL: n=7; M-II KO : n=6). k) . Quantification of different E0771 tumour-infiltrating T helper cells (IFNγ + Th1, IL4 + Th2 and IL17A + Th17) (n=11/group). l) . Quantification of E0771 tumour-infiltrating CD4 + -TL cells, macrophage, dendritic cells, B cells and neutrophils (CD45 + CD11b + Ly6G high ) (n=11/group). Data are presented as means ± s.e.m. The genetic backgrounds of mice are denoted with different colors shown on the right of (l) . WT, Tie2Cre and H2Ab floxP/floxP were combined as CTRL group. P values were calculated using two-tailed unpaired Student’s t-test (a–b, d–f, h–k) or two-tailed unpaired Mann–Whitney U test ( l ). n.s., not significant.

Article Snippet: The following antibodies against mouse antigens were used: anti-NG2 (1E6.4) and anti-EpCAM (caa7-9G8) (both from Miltenyi); anti-CD31 (MEC13.3), anti-CD45.1 (A20), anti-CD45.2 (104), anti-IFNγ (XMG1.2), anti-IL4 (11B11), anti-IL17A (TC11-18H10.1), anti-FoxP3 (MF14), anti-MHC-II (M5/114.15.2) (all from Biolegend); anti-CD25 (eBio7D4), anti-CD3ε (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-6.7) (all from eBioscience); anti-B220 (RA3-6B2), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD25 (PC61.5), anti-CD44 (IM7), anti-CD45 (30-F11), anti-CD62L (MEL14), anti-F4/80 (BM8) (all from Tonbo); anti-SiglecF (E50-2440, BD Biosciences).

Techniques: Flow Cytometry, Expressing, Suspension, Activation Assay, Two Tailed Test, MANN-WHITNEY

(A) Subcutaneous inoculation (s.c.) with 10 5 p.f.u. of VACV was administered to both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells, as well as CD25 + Foxp3 + Treg cells, was conducted in popliteal lymph nodes (pLN) at 6 and 14 days post-infection. The percentage of Th1 cells, CD8 CTL T cells, and Treg cells was assessed. The data represent means ± SEM from at least 8 mice per group, obtained from 3 independently conducted experiments, and were analyzed using unpaired Student’s t-test between genotypes at each time point. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant). (B) Intraperitoneal inoculation (i.p.) with 10 6 p.f.u. of VACV was performed on both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells was conducted in mesenteric lymph nodes (mLN) 6 days post-infection. The percentage of Th1 and CD8 CTL cells was determined. Data represent means ± SEM of at least 6 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is indicated by asterisks (*P < 0.05; **P < 0.01). (C) Infection with 10 5 p.f.u. of VACV through skin scarification (s.s.) in the tail was performed on WT and LysM-Lmna -/- mice. Flow cytometry analysis of the percentages of IFNγ-producing CD4 and CD8 T cells was conducted in extracted inguinal lymph nodes (iLN) at 6 and 14 days post-infection. (D) Viral load measurement in the tail skin was performed at 6 and 14 days post-infection. The data represent means ± SEM of at least 4 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant).

Journal: bioRxiv

Article Title: Role of Lamin A/C on dendritic cell function in antiviral immunity

doi: 10.1101/2024.05.14.593747

Figure Lengend Snippet: (A) Subcutaneous inoculation (s.c.) with 10 5 p.f.u. of VACV was administered to both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells, as well as CD25 + Foxp3 + Treg cells, was conducted in popliteal lymph nodes (pLN) at 6 and 14 days post-infection. The percentage of Th1 cells, CD8 CTL T cells, and Treg cells was assessed. The data represent means ± SEM from at least 8 mice per group, obtained from 3 independently conducted experiments, and were analyzed using unpaired Student’s t-test between genotypes at each time point. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant). (B) Intraperitoneal inoculation (i.p.) with 10 6 p.f.u. of VACV was performed on both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells was conducted in mesenteric lymph nodes (mLN) 6 days post-infection. The percentage of Th1 and CD8 CTL cells was determined. Data represent means ± SEM of at least 6 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is indicated by asterisks (*P < 0.05; **P < 0.01). (C) Infection with 10 5 p.f.u. of VACV through skin scarification (s.s.) in the tail was performed on WT and LysM-Lmna -/- mice. Flow cytometry analysis of the percentages of IFNγ-producing CD4 and CD8 T cells was conducted in extracted inguinal lymph nodes (iLN) at 6 and 14 days post-infection. (D) Viral load measurement in the tail skin was performed at 6 and 14 days post-infection. The data represent means ± SEM of at least 4 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant).

Article Snippet: Antibodies against: CD4 (Clone RM4-5, GK1.5) -v450, -APC and PE; CD25 (PC61.5) -APC; CD69 (H1.2F3)-FITC; CD28 (37.51); CD3 (145-2C11); CD45.1 (A20)-Pe-Cy7, -v450, -FITC; CD45.2 (104) -v450, -redFluor710; CD11b (M1/70)-v450; CD86 GL-1 PE; IFNγ XMG1.2 FITC, -APC; Foxp3 (3G3)-FITC; MHC-II (I-A/I-E, M5/115.15.2)-APC; GR1 (RB6-8C5)-Biotin; CD80 (16-10A1)-Biotin; B220 (RA3-6B2)-Biotin; F4/80 (BM8.1)-Biotin; CD11b (M1/70)-Biotin; CD19 (1D3)-Biotin; CD25 (PC61.5)-Biotin from Tonbo.

Techniques: Flow Cytometry, Infection, Derivative Assay

Flow cytometry was employed to assess the percentage of activated CD4 T cells, specifically gated on CD25 + or CD69 + cells; Th1-differentiated cells, specifically gated on IFNγ+ cells; and proliferating CellTrace Violet + T cells. Maturation of GM-CSF-derived WT- and LysM-Lmna -/- -BMDCs was undertaken with (A) sonicated VACV extract, (B) Pam3CSK4, or (C) lipopolysaccharide (LPS), and, in all conditions, pulsed with OVA323–339 cognate OT-IIp. (D) WT- or Lmna -/- -BMDCs, matured with LPS and pulsed with the OVA 323–339 cognate OT-II peptide, were s.c. inoculated into CD45.1 OT-II recipient mice. Six days later, pLNs were extracted, and IFNγ-producing CD4 T cells were analyzed by flow cytometry. The data represent means ± SEM and are presented from a representative experiment out of 3 (A and B, n=3) or out of 4 (C, n=4) or out of 2 (D, n=4). Activation data were analyzed using One Way ANOVA and Bonferroni post-test, and proliferation and Th1 differentiation were assessed by unpaired Student’s t-test. Statistical significance was denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant).

Journal: bioRxiv

Article Title: Role of Lamin A/C on dendritic cell function in antiviral immunity

doi: 10.1101/2024.05.14.593747

Figure Lengend Snippet: Flow cytometry was employed to assess the percentage of activated CD4 T cells, specifically gated on CD25 + or CD69 + cells; Th1-differentiated cells, specifically gated on IFNγ+ cells; and proliferating CellTrace Violet + T cells. Maturation of GM-CSF-derived WT- and LysM-Lmna -/- -BMDCs was undertaken with (A) sonicated VACV extract, (B) Pam3CSK4, or (C) lipopolysaccharide (LPS), and, in all conditions, pulsed with OVA323–339 cognate OT-IIp. (D) WT- or Lmna -/- -BMDCs, matured with LPS and pulsed with the OVA 323–339 cognate OT-II peptide, were s.c. inoculated into CD45.1 OT-II recipient mice. Six days later, pLNs were extracted, and IFNγ-producing CD4 T cells were analyzed by flow cytometry. The data represent means ± SEM and are presented from a representative experiment out of 3 (A and B, n=3) or out of 4 (C, n=4) or out of 2 (D, n=4). Activation data were analyzed using One Way ANOVA and Bonferroni post-test, and proliferation and Th1 differentiation were assessed by unpaired Student’s t-test. Statistical significance was denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant).

Article Snippet: Antibodies against: CD4 (Clone RM4-5, GK1.5) -v450, -APC and PE; CD25 (PC61.5) -APC; CD69 (H1.2F3)-FITC; CD28 (37.51); CD3 (145-2C11); CD45.1 (A20)-Pe-Cy7, -v450, -FITC; CD45.2 (104) -v450, -redFluor710; CD11b (M1/70)-v450; CD86 GL-1 PE; IFNγ XMG1.2 FITC, -APC; Foxp3 (3G3)-FITC; MHC-II (I-A/I-E, M5/115.15.2)-APC; GR1 (RB6-8C5)-Biotin; CD80 (16-10A1)-Biotin; B220 (RA3-6B2)-Biotin; F4/80 (BM8.1)-Biotin; CD11b (M1/70)-Biotin; CD19 (1D3)-Biotin; CD25 (PC61.5)-Biotin from Tonbo.

Techniques: Flow Cytometry, Derivative Assay, Sonication, Activation Assay

(A) 2C CD8 T cell proliferation to BALB/c stimulation was measured by CFSE dilution in MLRs set up as described in Methods. In some cases, MACS sorted splenic M-or G-MDSCs from B6 mice were added to the MLRs to determine their suppression of 2C T cell proliferation. In additional cases, rIFN-α (1000 U/ml) or anti-IFNaR1 (20 mg/ml) was further added to the cultures to determine their effects on the MDSC-mediated suppression of 2C proliferation. Representative histograms were gated on 2C CD8+ T cells. Percentage of proliferation in the right bar graph was calculated as (2C CD8+ T cells with diluted CFSE)/(total 2C CD8+ T cells). (B) B6 CD4+CD25+Foxp3+ Treg proliferation to BALB/c stimulation was measured by CFSE dilution in MLRs set up as described in Methods. In some cases, MACS sorted splenic M-or G-MDSCs from B6 mice were added to the MLRs to determine their effect on CD4+CD25+Foxp3+ Treg proliferation. In additional cases, rIFN-α or anti-IFNaR1 was further added to the cultures to determine their effects on the MDSC-mediated promotion of CD4+CD25+Foxp3+ Treg proliferation. Representative histograms were gated on CD4+CD25+Foxp3+ Tregs. Percentage of proliferation in the right bar graph was calculated as (CD4+CD25+Foxp3+ Tregs with diluted CFSE)/(total CD4+CD25+Foxp3+ Tregs). Data for (A-B) were obtained and averaged from three independent experiments. Statistical significance was determined by either two-way ANOVA with Bonferroni post-test or one-way ANOVA with Bonferroni post-test. *P ≤ 0.05, ***P ≤ 0.001.

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Receptor Tyrosine Kinase MerTK Suppresses an Allogenic Type I IFN Response to Promote Transplant Tolerance

doi: 10.1111/ajt.15087

Figure Lengend Snippet: (A) 2C CD8 T cell proliferation to BALB/c stimulation was measured by CFSE dilution in MLRs set up as described in Methods. In some cases, MACS sorted splenic M-or G-MDSCs from B6 mice were added to the MLRs to determine their suppression of 2C T cell proliferation. In additional cases, rIFN-α (1000 U/ml) or anti-IFNaR1 (20 mg/ml) was further added to the cultures to determine their effects on the MDSC-mediated suppression of 2C proliferation. Representative histograms were gated on 2C CD8+ T cells. Percentage of proliferation in the right bar graph was calculated as (2C CD8+ T cells with diluted CFSE)/(total 2C CD8+ T cells). (B) B6 CD4+CD25+Foxp3+ Treg proliferation to BALB/c stimulation was measured by CFSE dilution in MLRs set up as described in Methods. In some cases, MACS sorted splenic M-or G-MDSCs from B6 mice were added to the MLRs to determine their effect on CD4+CD25+Foxp3+ Treg proliferation. In additional cases, rIFN-α or anti-IFNaR1 was further added to the cultures to determine their effects on the MDSC-mediated promotion of CD4+CD25+Foxp3+ Treg proliferation. Representative histograms were gated on CD4+CD25+Foxp3+ Tregs. Percentage of proliferation in the right bar graph was calculated as (CD4+CD25+Foxp3+ Tregs with diluted CFSE)/(total CD4+CD25+Foxp3+ Tregs). Data for (A-B) were obtained and averaged from three independent experiments. Statistical significance was determined by either two-way ANOVA with Bonferroni post-test or one-way ANOVA with Bonferroni post-test. *P ≤ 0.05, ***P ≤ 0.001.

Article Snippet: The following Abs(clones) were used: Gr1-APC(RB6–8C5), Ly6C-PE-Cyanine7(HK1.4), Ly6G-PE(1A8); CD11b-APC-eFluor780(M1/70), CD11c-APC-eFluor780(N418), CD3e-PE-Cyanine7(145–2C11), CD3e-FITC(145–2C11), CD19-PerCP-Cyanine5.5(eBio1D3), CD4-PE(GK1.5), CD4-PE-Cyanine7(GK1.5), CD8a-APC-eFluor780(53–6.7), CD45-FITC(30-F11), CD25-eFluor450(PC61.5), F4/80-PerCP-Cyanine5.5(BM8), SIGN-R1-APC(eBio22D1), from eBioscience; CD68-FITC(FA-11), CD169-PE-Cyanine7(3D6.112), PD-L1-PerCP-Cyanine5.5(10F.9G2) from BioLegend; MerTK-PE or MerTK-APC(108928), ARG1-PE(Polyclonal) from R&D Systems; Foxp3-APC(3G3) from TONBO and CD3e-V500(500A2) from BD Biosciences.

Techniques:

(A) Heart allografts from Mertk−/− or Mertk+/+ recipients were retrieved on post-transplant day 10 and enumerated for CD4+CD25+Foxp3+ Tregs. Grafts from control untreated recipients are shown in the left dot plots. Grafts from recipients treated with donor (BALB/c) ECDI-SP are shown in the middle dot plots. Grafts from recipients further treated with anti-IFNaR1 as described in Methods are shown in the right dot plots. Representative dot blots were all gated on CD4+ cells. The bar graph on the right shows averaged total number of intra-graft Tregs from the various groups. (B) Heart allografts from Mertk−/− or Mertk+/+ recipients treated with BALB/c ECDI-SP were retrieved on post-transplant day 10, and graft-infiltrating G-MDSCs were analyzed for expression levels of Arginase-1 and PD-L1 by FACS. Representative histograms are shown on the left. The bar graph on the right shows the averaged MFI of Arginase-1 or PD-L1 of G-MDSCs in heart allografts from Mertk−/− or Mertk+/+ recipients. Data for (A-B) were obtained and averaged from a total of eight animals per group from three independent experiments. Statistical significance was determined using t test. *P ≤ 0.05, ***P ≤ 0.001.

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Receptor Tyrosine Kinase MerTK Suppresses an Allogenic Type I IFN Response to Promote Transplant Tolerance

doi: 10.1111/ajt.15087

Figure Lengend Snippet: (A) Heart allografts from Mertk−/− or Mertk+/+ recipients were retrieved on post-transplant day 10 and enumerated for CD4+CD25+Foxp3+ Tregs. Grafts from control untreated recipients are shown in the left dot plots. Grafts from recipients treated with donor (BALB/c) ECDI-SP are shown in the middle dot plots. Grafts from recipients further treated with anti-IFNaR1 as described in Methods are shown in the right dot plots. Representative dot blots were all gated on CD4+ cells. The bar graph on the right shows averaged total number of intra-graft Tregs from the various groups. (B) Heart allografts from Mertk−/− or Mertk+/+ recipients treated with BALB/c ECDI-SP were retrieved on post-transplant day 10, and graft-infiltrating G-MDSCs were analyzed for expression levels of Arginase-1 and PD-L1 by FACS. Representative histograms are shown on the left. The bar graph on the right shows the averaged MFI of Arginase-1 or PD-L1 of G-MDSCs in heart allografts from Mertk−/− or Mertk+/+ recipients. Data for (A-B) were obtained and averaged from a total of eight animals per group from three independent experiments. Statistical significance was determined using t test. *P ≤ 0.05, ***P ≤ 0.001.

Article Snippet: The following Abs(clones) were used: Gr1-APC(RB6–8C5), Ly6C-PE-Cyanine7(HK1.4), Ly6G-PE(1A8); CD11b-APC-eFluor780(M1/70), CD11c-APC-eFluor780(N418), CD3e-PE-Cyanine7(145–2C11), CD3e-FITC(145–2C11), CD19-PerCP-Cyanine5.5(eBio1D3), CD4-PE(GK1.5), CD4-PE-Cyanine7(GK1.5), CD8a-APC-eFluor780(53–6.7), CD45-FITC(30-F11), CD25-eFluor450(PC61.5), F4/80-PerCP-Cyanine5.5(BM8), SIGN-R1-APC(eBio22D1), from eBioscience; CD68-FITC(FA-11), CD169-PE-Cyanine7(3D6.112), PD-L1-PerCP-Cyanine5.5(10F.9G2) from BioLegend; MerTK-PE or MerTK-APC(108928), ARG1-PE(Polyclonal) from R&D Systems; Foxp3-APC(3G3) from TONBO and CD3e-V500(500A2) from BD Biosciences.

Techniques: Control, Expressing