Journal:

Article Title: Induction of Specific Cytotoxic Lymphocytes in Mice Vaccinated with Brucella abortus RB51

doi: 10.1128/IAI.69.9.5502-5508.2001

Figure Lengend Snippet: Different roles of CD4+ and CD8+ T cells in the cytotoxic lysis and release of IFN-γ. The two T-cell populations were isolated by the MACS magnetic kit and showed more than 92% purity by flow cytometry. The individual T-cell populations were cocultured at various concentrations with either strain RB51-infected or normal J774.A1 cells. The cytotoxic activity (A) and the amount of IFN-γ released into the supernatants (B) were measured. The data are means for triplicate estimations, and standard deviations did not exceed 20% of the means. Cocultures of CD4+ T cells with noninfected target macrophages (solid squares) and RB51-pulsed target macrophages (solid triangles) and CD8+ T cells with noninfected target cells (solid diamonds) and RB51-pulsed target cells (solid circles) were tested.

Article Snippet: Briefly, live T cells isolated by Histopaque column purification were incubated with MACS magnetic MicroBeads to which monoclonal antibodies against CD4 molecule (clone GK1.5; isotype, rat IgG2b) or CD8 molecule (clone 53-6.7; isotype, rat IgG2a) had been coupled (Miltenyi Biotec, Auburn, Calif.) at the concentration of 10 μl of MicroBeads per 10 7 total cells for 15 min in a refrigerator at 4°C (see Fig. ).

Techniques: Lysis, Isolation, Flow Cytometry, Infection, Activity Assay

Journal:

Article Title: Induction of Specific Cytotoxic Lymphocytes in Mice Vaccinated with Brucella abortus RB51

doi: 10.1128/IAI.69.9.5502-5508.2001

Figure Lengend Snippet: Specific phenotype analysis of effector cells by flow cytometry a

Article Snippet: Briefly, live T cells isolated by Histopaque column purification were incubated with MACS magnetic MicroBeads to which monoclonal antibodies against CD4 molecule (clone GK1.5; isotype, rat IgG2b) or CD8 molecule (clone 53-6.7; isotype, rat IgG2a) had been coupled (Miltenyi Biotec, Auburn, Calif.) at the concentration of 10 μl of MicroBeads per 10 7 total cells for 15 min in a refrigerator at 4°C (see Fig. ).

Techniques: Flow Cytometry

Journal: Nature Communications

Article Title: Fasting mimicking diet in mice delays cancer growth and reduces immunotherapy-associated cardiovascular and systemic side effects

doi: 10.1038/s41467-023-41066-3

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD4, VioBright FITC (REA604) , Miltenyi Biotec , 130-118-692.

Techniques: In Vivo

Journal: Science Advances

Article Title: Altered X-chromosome inactivation predisposes to autoimmunity

doi: 10.1126/sciadv.adn6537

Figure Lengend Snippet: ( A ) Representative flow cytometry analysis of spontaneously activated B220 + CD69 + B cells in spleen from 1-year-old WT and Ftx −/− females. Percentages in leucocytes are shown on the graphs beneath. Each triangle represents a mouse. Median values are shown (Mann-Whitney test, * P < 0.05 and ** P < 0.01). ( B ) Same as (A) for spontaneously activated CD4 + CD69 + T cells (Mann-Whitney test, * P < 0.05 and ** P < 0.01). ( C ) Total IgG, IgM, IgG2b, and IgG2c natural antibody levels in sera of 3-month-, 1-year, and >1.5-year-old WT or Ftx −/− females measured by ELISA. Each circle represents a mouse. Mean values are shown (Mann-Whitney test, * P < 0.05 and ** P < 0.01). ( D ) Cytokines levels in the blood analyzed with cytometric bead array assays on sera from 3-month-, 1-year-, or 2-year-old WT and Ftx −/− females. Each triangle represents a mouse. Median values are shown ( t test, * P < 0.05).

Article Snippet: BM, spleen, blood, and peritoneal cavity cells were stained using the following antibodies: CD3 PerCP-Vio770 (130-119-656, Miltenyi Biotec), CD4-allophycocyanin (APC) (130-123-207, Miltenyi Biotec), CD5-APC-Vio770 (130-120-165, Miltenyi Biotec), CD8-fluorescein isothiocyanate (FITC) (130-118-468, Miltenyi Biotec), CD11b APC (553312, BD Pharmingen), CD11c phycoerythrin (PE)–Vio770 (130-110-840, Miltenyi Biotec), CD19-FITC (557398, BD Pharmingen), CD21-APC-Vio770 (130-111-733, Miltenyi Biotec), CD23-PE-Vio770 (130-118-764, Miltenyi Biotec), CD38-PE (130-123-571, Miltenyi Biotec), CD43-PE (130-112-887, Miltenyi Biotec), CD69-PE (130-115-575, Miltenyi Biotec), CD138 PE-Vio615 (130-108-989, Miltenyi Biotec), F4/80 FITC (130-117-509, Miltenyi Biotec), Ter119 PE (130-112-909, Miltenyi Biotec), SiglecH APC-Vio770 (130-112-299, Miltenyi Biotec), B220-APC (130-110-847, Miltenyi Biotec), B220 VioBlue (130-110-851, Miltenyi Biotec), IgM-VioBlue (130-116-318, Miltenyi Biotec), IgD-PE (130-111-496, Miltenyi Biotec), GL7-PE-Cy7 (144619, BioLegend), Ly6C-FITC (130111-915, Miltenyi Biotec), streptavidin FITC (554060, BD Biosciences), CD138 BV605 (563147, BD-Horizon), CD23 BV605 (101637, BioLegend), I-A/I-E BV711 (107643, BioLegend), CD19 BV786 (563333, BD Horizon), T and B cell activation antigen (GL7) PE (561530, BD Pharmingen), CD95 PE-Cy7 (557653, BD Pharmingen), IgM APC-eFluor 780 (47-5790-82, Invitrogen), CD45R/B220 APC/cyanine7 (103224, BioLegend), CD11b eFluor 450 (48-0112-82, eBioscience), CD267 (TACI) BV421 (742840, BD Biosciences), IgD BUV395 (564274, BD Horizon), streptavidin APC (4317-82, eBioscience), CD3 Biotin (100304, BioLegend), Biotin CD11c (568970, BD Biosciences), CD21 PercP Cy5.5 (562797, BD Biosciences), and Fixable Viability Dye eFluor 506 (65-0866-18, Invitrogen) following the recommendations of the manufacturers.

Techniques: Flow Cytometry, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: DNase I alleviates renal inflammatory injury in MRL/lpr mice by inhibiting NETs formation

doi: 10.3389/fimmu.2025.1656069

Figure Lengend Snippet: DNase I treatment decreased the number of immune inflammatory cells in the kidneys and spleens of mice in the MRL/lpr model. (A, B) The TIMER algorithm was used to assess the proportion of immune cell infiltration in the kidneys of control (Ctr), MRL/lpr (Lpr), and DNase I-treated MRL/lpr (DNase) mice at 21 weeks of age. The percentages of CD3+CD4+ T cell subsets (C, G) , CD3+CD8+ T cell subsets (D, H) , and CD3-CD19+B cell subsets (E, I) in the spleen were quantified using flow cytometry across the normal control, MRL/lpr, and DNase I treatment MRL/lpr group. (F, J) Flow cytometric and quantitative analysis of CD4+CD25+Foxp3+ regulatory T cell (Treg) percentages in the spleens of mice from each experimental group were also conducted. Data were expressed as means ± SD for groups of three mice. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. normal control ( t test). # P < 0.05 vs. MRL/lpr mice (Bonferroni correction; two comparisons were made).

Article Snippet: The following antibodies were utilized for staining: anti-CD3 antibody (E-AB-F1013Q), anti-CD4 antibody (E-AB-F1097S), anti-CD8 antibody (E-AB-F1104J), anti-CD19 antibody (E-AB-F0986E), anti-CD25 antibody (E-AB-F1102D), and anti-Foxp3 antibody (E-AB-F1238E), all sourced from Elabscience (China).

Techniques: Control, Flow Cytometry

Journal: Advanced Science

Article Title: CXCL13 Expression Promotes CAR T Cell Antitumor Activity and Potentiates Response to PD‐1 Blockade

doi: 10.1002/advs.202508095

Figure Lengend Snippet: CXCL13 is highly expressed in T cells from responders to ICB therapy. a). UMAP plot displaying 237231 CD3 + T cells across 8 cancer types: BCC (basal cell carcinoma, n = 11 patients; n = 22 samples), SCC (squamous cell carcinoma, n = 3 patients; n = 8 samples), BLCA (bladder cancer, n = 6 patients; n = 6 samples), TNBC (triple‐negative breast cancer, n = 8 patients; n = 14 samples), RCC (Renal cell carcinoma, n = 7 patients; n = 7 samples), HNSCC (head and neck cancer, n = 4 patients; n = 8 samples), NSCLC (non‐small‐cell lung cancer, n = 36 patients; n = 47 samples), and PRAD (prostate cancer, n = 10 patients; n = 12 samples), categorized into 13 distinct cell types. b). Comparison of differential genes in CD3 + , CD8 + , and CD4 + T cells in the response and non‐response groups. c,d). UMAP plots (c) and dot plots (d) showing the expression of CXCL13 in CD3 + , CD8 + , and CD4 + T cells of pre‐treatment non‐response, pre‐treatment response, post‐treatment non‐response, and post‐treatment response groups. e). CXCL13 expression levels in responders and non‐responders across multiple tumor cohorts treated with ICB: HNSC, melanoma cohort 1 (anti‐PD‐1 and anti‐CTLA‐4), melanoma cohort 2 (anti‐PD‐1), STAD (stomach adenocarcinoma), and BLCA. f). Comparison of CXCL13 expression in pre‐treatment and post‐treatment groups from melanoma cohort 1 and melanoma cohort 2 treated with ICB. g). Kaplan‐Meier survival curves stratified by high and low CXCL13 expression in ICB‐treated patients from BLCA, melanoma cohort 1, and melanoma cohort 2. h). Heatmap showing the correlation of CXCL13 expression with various cell types across tumor types in the TCGA dataset. Cell‐type deconvolution was performed using TIMER2.0, with significant correlations ( p < 0.05) marked by solid indicators.

Article Snippet: The following antibodies were used for staining: anti‐mouse CD45.1, BV450 (E‐AB‐F1184UQ), Elabscience, Cat # AF19855; anti‐mouse CD45.1, PE (A20), Biolegend, Cat # 110 708; anti‐mouse CD3ε, PE/Cyanine7 (145‐2C11), Biolegend, Cat # 100 320; anti‐mouse PD‐1, PE/Cyanine7 (RMP1‐30), Biolegend, Cat # 109 110; anti‐mouse CD4, APC/Cy7 (E‐AB‐F1353UJ), Elabscience, Cat # AF18028; anti‐mouse CD4, PerCP/Cyanine5.5 (RM4‐5), Biolegend, Cat # 100 540; anti‐mouse CD4, PE/Cyanine7 (RM4‐5), Biolegend, Cat # 100 527; anti‐mouse CD4, Brilliant Violet 785 (RM4‐5), Biolegend, Cat # 100 552; anti‐mouse CD8a, PE (53‐6.7), Biolegend, Cat # 100 708; anti‐mouse CD8a, APC (53‐6.7), Biolegend, Cat # 100 712; anti‐mouse CD8a, PE/Cyanine7 (53‐6.7), Biolegend, Cat # 100 721; anti‐mouse CD45, PerCP/Cyanine5.5 (30‐F11), Biolegend, Cat # 103 132; anti‐mouse Ly108, PE (330‐AJ), Biolegend, Cat # 134 605; anti‐mouse LAG‐3, PerCP/Cyanine5.5 (C9B7W), Biolegend, Cat # 125 211; anti‐mouse LAG‐3, APC (C9B7W), Biolegend, Cat # 125 210; anti‐mouse CD69, Brilliant Violet 605 (H1.2F3), Biolegend, Cat # 104 530; anti‐mouse/human CD44, Brilliant Violet 510 (IM7), Biolegend, Cat # 103 044; anti‐mouse CD62L, PerCP/Cyanine5.5 (MEL‐14), Biolegend, Cat # 104 432; anti‐mouse CTLA‐4, APC (UC10‐4B9), Biolegend, Cat # 106 309; anti‐human CD3, APC (OKT3), Biolegend, Cat # 317 318; anti‐human CD4, PerCP/Cyanine5.5 (RPA‐T4), Biolegend, Cat # 300 530; anti‐human CD8, APC/Cyanine7 (SK1), Biolegend, Cat # 344 713; anti‐human CD279(PD‐1), PE (A17188A), Biolegend, Cat # 379 209; anti‐human CD366(Tim3), PE/CY7 (F38‐2E2), Biolegend, Cat # 345 014.

Techniques: Comparison, Expressing

Journal: Advanced Science

Article Title: CXCL13 Expression Promotes CAR T Cell Antitumor Activity and Potentiates Response to PD‐1 Blockade

doi: 10.1002/advs.202508095

Figure Lengend Snippet: CXCL13 CAR T cells display enhanced antitumor characteristics. a). GSEA analysis of the mitochondrial membrane potential gene set in bulk RNA‐seq comparing CXCL13 CAR T cells with control CAR T cells. b–d). Flow cytometry analysis of mitochondrial membrane potential (TMRM) in control CAR T and CXCL13 CAR T cells. Representative TMRM peak plots are shown for GFP + (b) and GFP − (c) T cells cultured in vitro on day 10 and for cells isolated from the spleen (d). TMRM: Tetramethylrhodamine, methyl ester. Mean ± SEM, n = 3 (b, c); n = 5 (control CAR T), n = 4 (CXCL13 CAR T) (d). e). Intracellular ATP levels in control CAR T cells and CXCL13 CAR T cells. Mean ± SEM, n = 4. f,g). GSEA analysis of positive regulation of T cell migration gene set in CXCL13 CAR T cells versus control CAR T cells using bulk RNA‐seq data (f) and CXCL13 + cells versus CXCL13 − cells using scRNA‐seq data (g). h). Schematic overview of the transwell migration assay. i,j). The migration ability of CAR T cells was assessed by the transwell assay, showing the proportion (i) and number (j) of cells that migrated through the membrane. Mean ± SEM, n = 4. k–m). C57BL/6 mice injected s.c. with 5 × 10 5 B16‐CD19 cells were treated with 2×10⁶ CAR T cells on day 5 post‐tumor establishment. The number of CAR T cells was detected after 7 days of adoptive transfer in the spleen (k), draining lymph nodes (l), and tumor sites (m). Mean ± SEM, n = 3. n). Percentage of CD4 + T cells among CD3 + T cells from patients with GPC3‐positive solid tumors treated with CAR T cells (non‐responders [NR], n = 6; responders [R], n = 15). Mean ± SEM. o,p). Flow cytometry plots (o) and line graphs (p) showing the percentage of CD4⁺ CAR T cells in vitro from day 4 to day 10. Mean ± SEM, n = 4. q–s). Proportions of CD4 + and CD8 + CAR T cells in blood (q), spleen (r), and draining lymph nodes (s). Mean ± SEM, blood ( n = 6), spleen ( n = 6 for control CAR T; n = 4 for CXCL13 CAR T), draining lymph nodes ( n = 6 for control CAR T; n = 4 for CXCL13 CAR T). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; two‐tailed unpaired t‐test (b–e,i–n,p–s).

Article Snippet: The following antibodies were used for staining: anti‐mouse CD45.1, BV450 (E‐AB‐F1184UQ), Elabscience, Cat # AF19855; anti‐mouse CD45.1, PE (A20), Biolegend, Cat # 110 708; anti‐mouse CD3ε, PE/Cyanine7 (145‐2C11), Biolegend, Cat # 100 320; anti‐mouse PD‐1, PE/Cyanine7 (RMP1‐30), Biolegend, Cat # 109 110; anti‐mouse CD4, APC/Cy7 (E‐AB‐F1353UJ), Elabscience, Cat # AF18028; anti‐mouse CD4, PerCP/Cyanine5.5 (RM4‐5), Biolegend, Cat # 100 540; anti‐mouse CD4, PE/Cyanine7 (RM4‐5), Biolegend, Cat # 100 527; anti‐mouse CD4, Brilliant Violet 785 (RM4‐5), Biolegend, Cat # 100 552; anti‐mouse CD8a, PE (53‐6.7), Biolegend, Cat # 100 708; anti‐mouse CD8a, APC (53‐6.7), Biolegend, Cat # 100 712; anti‐mouse CD8a, PE/Cyanine7 (53‐6.7), Biolegend, Cat # 100 721; anti‐mouse CD45, PerCP/Cyanine5.5 (30‐F11), Biolegend, Cat # 103 132; anti‐mouse Ly108, PE (330‐AJ), Biolegend, Cat # 134 605; anti‐mouse LAG‐3, PerCP/Cyanine5.5 (C9B7W), Biolegend, Cat # 125 211; anti‐mouse LAG‐3, APC (C9B7W), Biolegend, Cat # 125 210; anti‐mouse CD69, Brilliant Violet 605 (H1.2F3), Biolegend, Cat # 104 530; anti‐mouse/human CD44, Brilliant Violet 510 (IM7), Biolegend, Cat # 103 044; anti‐mouse CD62L, PerCP/Cyanine5.5 (MEL‐14), Biolegend, Cat # 104 432; anti‐mouse CTLA‐4, APC (UC10‐4B9), Biolegend, Cat # 106 309; anti‐human CD3, APC (OKT3), Biolegend, Cat # 317 318; anti‐human CD4, PerCP/Cyanine5.5 (RPA‐T4), Biolegend, Cat # 300 530; anti‐human CD8, APC/Cyanine7 (SK1), Biolegend, Cat # 344 713; anti‐human CD279(PD‐1), PE (A17188A), Biolegend, Cat # 379 209; anti‐human CD366(Tim3), PE/CY7 (F38‐2E2), Biolegend, Cat # 345 014.

Techniques: Membrane, RNA Sequencing, Control, Flow Cytometry, Cell Culture, In Vitro, Isolation, Migration, Transwell Migration Assay, Transwell Assay, Injection, Adoptive Transfer Assay, Two Tailed Test