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: Frontiers in immunology

Article Title: IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities.

doi: 10.3389/fimmu.2023.1185618

Figure Lengend Snippet: FIGURE 1 Downregulation of IRF4 expression in CAR T cells. (A) Schematic of CAR constructs. (B) CAR expression in T cells detected by staining with a phycoerythrin (PE)-labeled goat anti-human IgG antibody before (upper panels) and after (lower panels) magnetic cell separation (MACS). One representative donor out of four is shown. (C) Intracellular staining of IRF4 after stimulation with CEA+ BxPC-3 cells at the indicated time points in CD8+ (left panel) and CD4+ CAR T cells (right panel). CAR T cells were generated as described in the materials and methods section by activation of PBMCs followed by retroviral transduction. Untransduced cells were generated by activation of PBMCs and subsequent expansion with IL-2, but without retroviral transduction. Data represent means ± SEM of five donors, p values were calculated by Student´s t test, *indicates p ≤0.05, ***indicates p ≤0.001.

Article Snippet: Four hours later, T cells were stained with the viability dye eFluor 780, the goat F(ab’)2 antihuman IgG-PE antibody, a BV421-conjugated anti-CD8 antibody (BD), and an APC-conjugated anti-CD4 antibody (Miltenyi Biotech).

Techniques: Expressing, Construct, Staining, Labeling, Magnetic Cell Separation, Generated, Activation Assay, Retroviral, Transduction

Journal: Frontiers in immunology

Article Title: IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities.

doi: 10.3389/fimmu.2023.1185618

Figure Lengend Snippet: FIGURE 3 Downregulation of IRF4 enhances CAR T cell functionality. (A) CAR T cells (starting with 1 × 105 CAR T cells) underwent four rounds (R1-R4) of stimulation with GFP-labeled CEA+ BxPC-3 cells (1 × 105 tumor cells at the beginning of each round). At the end of each round, CAR T cells (live CD3+ CAR+) (left panel) and BxPC-3 cells (right panel) were quantified by flow cytometry. Data represent means ± SEM of six donors, p values were calculated by Student´s t test, ns indicates not significant, and * indicates p ≤0.05. (B-G) Phenotypic analysis of CD8+ CAR T cells during repetitive antigen stimulation. CAR T cells underwent three rounds (R1-R3) of antigen-stimulation with unlabeled BxPC-3 cells. At the end of each round, the CD8/CD4 T cell ratio was determined (B). CAR T cells were stained for CD8 and further characterized regarding TIM-3 (C), PD-1 (D), TIGIT (E) expression and effector-memory cell differentiation: SCM = T stem-cell-memory (CD45RO+ CD62L+), EM = effector-memory (CD45RO+ CD62L-), CM = central-memory (CD45RO+ CD62L+), E = effector (CD45RO- CD62L-) (F), and CD27 expression (G). Data represent geometric means of ± SEM of at least four donors, p values were calculated by paired t test, ns indicates not significant, *indicates p ≤0.05, **indicates p ≤0.01.

Article Snippet: Four hours later, T cells were stained with the viability dye eFluor 780, the goat F(ab’)2 antihuman IgG-PE antibody, a BV421-conjugated anti-CD8 antibody (BD), and an APC-conjugated anti-CD4 antibody (Miltenyi Biotech).

Techniques: Labeling, Cytometry, Staining, Expressing, Cell Differentiation

Journal: Translational oncology

Article Title: Tumor-specific T cells in head and neck cancer have rescuable functionality and can be identified through single-cell co-culture.

doi: 10.1016/j.tranon.2024.101899

Figure Lengend Snippet: Fig. 1. Comparison of exhaustion marker expression (top panels) and memory marker expression (bottom panels) from flow cytometry of freshly digested tumor biopsies or patient-matched circulating T cells for CD4 and CD8 subsets. ***p<0.001, **p<0.01. Exhaustion markers assessed in n = 57 samples (n = 29 TIL and n = 28 circulating T cell). Memory markers assessed in n = 52 samples (n = 24 TIL and n = 28 circulating T cell).

Article Snippet: For immunophenotyping of digested HNSCC specimens, the following antibodies were used: VioBlue antihuman CD223 (Miltenyi, clone REA351), VioGreen anti-human CD4 (Miltenyi, clone M-T466), FITC anti-human CD45 (Miltenyi, clone 5B1), PE anti-human TIGIT (Miltenyi, clone REA 1202), PE-Vio770 antihuman CD279 (Miltenyi, clone REA1165), APC anti-human TIM3 (Miltenyi, clone F38–2E2), APC-Vio770 anti-human (Miltenyi, clone REA734), VioBlue anti-human CD45RA (Miltenyi, clone REA562), FITC anti-human CD3 (Miltenyi, clone REA613), PE-Vio770 anti-human CD62L (Miltenyi, clone REA615), APC anti-human CD45RO (Miltenyi, clone REA611).

Techniques: Comparison, Marker, Expressing, Flow Cytometry

Journal: Translational oncology

Article Title: Tumor-specific T cells in head and neck cancer have rescuable functionality and can be identified through single-cell co-culture.

doi: 10.1016/j.tranon.2024.101899

Figure Lengend Snippet: Fig. 2. Comparison of exhaustion marker expression (top panels) and memory marker expression (bottom panels) from flow cytometry of expanded TILs or patient- matched circulating T cells at early (day 4–5 post-activation) and late (day 10–11 post-activation) for CD4 and CD8 subsets. ***p<0.0001, +p = 0.038, *p = 0.050. Exhaustion markers assessed in n = 6 samples (n = 3 TIL and n = 3 circulating T cell). Memory markers assessed in n = 4 samples (n = 2 TIL and n = 2 circulating T cell).

Article Snippet: For immunophenotyping of digested HNSCC specimens, the following antibodies were used: VioBlue antihuman CD223 (Miltenyi, clone REA351), VioGreen anti-human CD4 (Miltenyi, clone M-T466), FITC anti-human CD45 (Miltenyi, clone 5B1), PE anti-human TIGIT (Miltenyi, clone REA 1202), PE-Vio770 antihuman CD279 (Miltenyi, clone REA1165), APC anti-human TIM3 (Miltenyi, clone F38–2E2), APC-Vio770 anti-human (Miltenyi, clone REA734), VioBlue anti-human CD45RA (Miltenyi, clone REA562), FITC anti-human CD3 (Miltenyi, clone REA613), PE-Vio770 anti-human CD62L (Miltenyi, clone REA615), APC anti-human CD45RO (Miltenyi, clone REA611).

Techniques: Comparison, Marker, Expressing, Flow Cytometry, Activation Assay

Journal: Translational oncology

Article Title: Tumor-specific T cells in head and neck cancer have rescuable functionality and can be identified through single-cell co-culture.

doi: 10.1016/j.tranon.2024.101899

Figure Lengend Snippet: Fig. 4. After TIL and circulating T cell isolation, T cells from P012 were rested overnight and underwent evaluation with IsoLight single-cell proteomics immediately after CD3/CD28 activation. CD8 and CD4 TILs secreted effector proteins to a greater extent than patient-matched CD8 and CD4 circulating T cells. In particular, CD8 TILs demonstrated polyfunctional secretion of both effector and chemoattractive proteins.

Article Snippet: For immunophenotyping of digested HNSCC specimens, the following antibodies were used: VioBlue antihuman CD223 (Miltenyi, clone REA351), VioGreen anti-human CD4 (Miltenyi, clone M-T466), FITC anti-human CD45 (Miltenyi, clone 5B1), PE anti-human TIGIT (Miltenyi, clone REA 1202), PE-Vio770 antihuman CD279 (Miltenyi, clone REA1165), APC anti-human TIM3 (Miltenyi, clone F38–2E2), APC-Vio770 anti-human (Miltenyi, clone REA734), VioBlue anti-human CD45RA (Miltenyi, clone REA562), FITC anti-human CD3 (Miltenyi, clone REA613), PE-Vio770 anti-human CD62L (Miltenyi, clone REA615), APC anti-human CD45RO (Miltenyi, clone REA611).

Techniques: Cell Isolation, Activation Assay

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