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: Journal of Experimental & Clinical Cancer Research : CR

Article Title: SKP2 drives the sensitivity to neddylation inhibitors and cisplatin in malignant pleural mesothelioma

doi: 10.1186/s13046-022-02284-7

Figure Lengend Snippet: MLN4924 in combination with cisplatin effectively restored immunogenic cell death in resistant mesothelioma cells. Primary MPM cells derived from 3 different histopathological subtypes, epithelioid (EPI BAP1 + UPN7 and EPI BAP1 - UPN11), biphasic (BIP BAP1 + UPN14 and BIP BAP1 - UPN16) and sarcomatous (SAR BAP1 + UPN22 and SAR BAP - UPN23) were incubated in fresh medium (CTRL), with 50 μM cisplatin (PT), 0.2 μM MLN4924 (MLN) or their combination (PT+MLN) for 24 h Panels A-B) or 48 h (panel C ). A . Calreticulin (CRT) exposure on the plasma membrane was measured by flow cytometry in duplicates. Data are expressed as means of 2 primary MPM for each histopathological subtype used + SD ( n = 3). *** p < 0.001: treated cells vs CTRL cells; ° p < 0.05, °°° p < 0.001: PT+MLN-treated cells vs PT-treated cells. B . ATP release was measured by a chemiluminescence-based assay in duplicates. Data are expressed as means of 2 primary MPM for each histopathological subtype used + SD ( n = 3). *** p < 0.001: treated cells vs CTRL cells; °°° p < 0.001: PT+MLN-treated cells vs PT-treated cells. C . HMGB1 release was measured by ELISA in duplicates. Data are expressed as means of 2 primary MPM for each histopathological subtype used + SD ( n = 3). ** p < 0.01, *** p < 0.001: treated cells vs CTRL cells; °° p < 0.01: PT+MLN-treated cells vs PT-treated cells. D . Phagocytosis by dendritic cells was measured by flow cytometry in duplicates. Data are expressed as means of 2 primary MPM for each histopathological subtype used + SD ( n = 3). * p < 0.05, *** p < 0.001: treated cells vs CTRL cells; ° p < 0.05, °°° p < 0.001: PT+MLN-treated cells vs PT-treated cells. E . CD8 + CD107a + lymphocytes, collected after co-culture with dendritic cells that have phagocytized MPM cells, were quantified by flow cytometry, in duplicates. Data are expressed as means of 2 primary MPM for each histopathological subtype used+ SD ( n = 3). *** p < 0.001: treated cells vs CTRL cells; °°° p < 0.01: PT+MLN-treated cells vs PT-treated cells. F-G . AB1 tumors were subcutaneously implanted into 6-week-old female BALB/c mice when the tumor size reached 50 mm 3 mice ( n = 10 animals/group) were treated for 3 consecutive as indicated in the Methods section. F . Percentage of CD4 + /CD8 + intratumor T-lymphocytes (TILs) collected after tumor digestion and analyzed by flow cytometry, in duplicates. Data are means + SD. *** p < 0.001: treated group vs CTRL group; °°° p < 0.001: PT+MLN-treated cells vs PT-treated cells. G . Representative immunohistochemical imaging of CD4 + and CD8 + TILs. Scale bar: 100 μm. (10x ocular, 40x objective). At least 10 fields were examined for each condition

Article Snippet: Tumor infiltrating lymphocytes (TILs) were isolated from 2×10 6 single-cell suspension derived from the homogenated tumors, using the CD8 (TIL) MicroBeads (mouse, 130-116-478, Miltenyi Biotec., Bergisch Gladbach, Germany) and the CD4 (TIL) MicroBeads (mouse, 130-116-475, Miltenyi Biotec.), in order to obtained a purified population of TILs that represent a small percentage of cells contained in the tumor mass, ready for the quantification by flow cytometry.

Techniques: Derivative Assay, Incubation, Clinical Proteomics, Membrane, Flow Cytometry, Chemiluminescence Immunoassay, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Immunohistochemical staining, Imaging