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