flow cytometry analysis  (Miltenyi Biotec)

Journal: International immunopharmacology

Article Title: HMGB1 promotes neutrophil PD-L1 expression through TLR2 and mediates T cell apoptosis leading to immunosuppression in sepsis.

doi: 10.1016/j.intimp.2024.112130

Figure Lengend Snippet: Fig. 5. In sepsis, HMGB1 and TLR2 regulate the expression of PD-1 on T lymphocytes to mediate the occurrence of immune suppression. (A) Representative double- staining immunofluorescence of T Lymphocytes (CD3) with PD-1 in Sham, CLP, and CLP + GA mice (n = 3, scale = 50 μm). (B) Flow sorting of spleen T lymphocytes (n = 3). (C) Western blot compared the expression of PD-1 on T lymphocytes in sham, CLP and CLP + GA groups (n = 3). (D) Representative double-staining immunofluorescence of T Lymphocytes (CD3) with PD-1 in Sham, CLP and CLP + C29 mice (n = 3, scale = 100 μm). (E) Flow sorting of spleen T lymphocytes (n = 3). (F) Western blot compared the expression of PD-1 on T lymphocytes in sham, CLP and CLP + C29 groups (n = 4). Glycyrrhizic acid (GA) is an HMGB1 inhibitor; C29 is a TLR2 inhibitor. Data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ns means no significance.

Article Snippet: T lymphocytes sorted by APC anti-Mouse CD3 flow cytometry was lysed with RIPA lysis buffer (GBCBIO) containing 1 mM PMSF (Ubio), 1 × phosphatase inhibitor cocktail (GLPBIO), and 1 × EDTA-free protease inhibitor cocktail (TargetMol).

Techniques: Expressing, Double Immunofluorescence Staining, Western Blot

Journal: iScience

Article Title: The immune checkpoint ICOSLG is a relapse-predicting biomarker and therapeutic target in infant t(4;11) acute lymphoblastic leukemia

doi: 10.1016/j.isci.2022.104613

Figure Lengend Snippet:

Article Snippet: Mouse Anti-Human CD3, monoclonal antibody, APC-H7 conjugated (flow cytometry) , BD , BD Biosciences Cat# 560176, RRID: AB_1645475.

Techniques: Recombinant, Flow Cytometry, Neutralization, Functional Assay, Western Blot, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software, Fluorescence, Marker

Journal: Frontiers in Immunology

Article Title: Human T2R38 Bitter Taste Receptor Expression in Resting and Activated Lymphocytes

doi: 10.3389/fimmu.2018.02949

Figure Lengend Snippet: Expression of T2R38 receptor in human PBMC. (A) Age-related T2R38 expression in PBMC from young (20–35 years, n ≥ 10) and elderly (60–90 years, n = 9) subjects. A representative staining from one person is shown for each cell type; T2R38 (continuous line) and rabbit IgG (dotted line). (B) T2R38 expression in CD3+ T cells from young ( n = 7) and elderly ( n = 7) donors (C) Sex difference of T2R38 expression in lymphocytes from male and female donors ( n = 10). (D) T2R38 expression in CD3-PE stained T cells and CD19-APC stained B cells from human volunteers, ( n = 6). A representative staining from one person is shown; CD3+ (continuous line) and CD19+ (dotted line). (E) Haplotype analysis was done using amplification of a TAS2R38 864bp fragment followed by sequencing. PAV: p roline, a lanine and v aline; AVI: a lanine, v aline and i soleucine ( n = 19). T2R38 expression (delta MESF) was quantified by Quantum Alexa Fluor 488 MESF using cytofluorometry (Supplementary Figure ), IgG was used as isotype control. Each dot presents one donor in the graphs. The gating strategy is shown in Supplementary Figure . Significance of difference was calculated relatively to the respective control, * p < 0.05, ** p < 0.01.

Article Snippet: The following primary human antibodies labeled with fluorophore were used for flow cytometry: CD3-APC (clone BW264/56), CD3-PE (clone REA613), CD4-PE (clone 15E8), CD4-APC (clone REA 623), CD8-PE (clone BW135/80), CD8-APC (clone BW135/80), CD45RO-Percp (clone UCHL1), CD62L-APC (Clone 145/15), CD69-PE (Clone FN50), CD11b (clone M1/70.15.11.5), CD14-PE (clone REA599) from Miltenyi Biotec (Bergisch Gladbach, Germany), CD19-APC (clone HIB19) from eBioscience, CD25-Percp (clone M-A251) from Bioledgen.

Techniques: Expressing, Staining, Amplification, Sequencing, Control

Journal: Frontiers in Immunology

Article Title: Human T2R38 Bitter Taste Receptor Expression in Resting and Activated Lymphocytes

doi: 10.3389/fimmu.2018.02949

Figure Lengend Snippet: Effect of activation on T2R38 expression in PBMC. (A) PBMC were stimulated with different concentrations of PMA/ionomycin (1 μg/ml), PHA or anti-CD3/CD28 mAbs for 72 h ( n ≥ 5) (B) Isolated PBMC from young and elderly individuals were stimulated with 1 ng/ml CD3/CD28 for 72 h ( n ≥ 5) (C) PBMC were stimulated with 1 ng/ml CD3/CD28 for the indicated time points, activated cells were determined by the percentage of blast cells ( n ≥ 3) (D) PBMC were stimulated with 1 ng/ml CD3/CD28 and T2R38 expression on CD69+/CD25+ CD3+T lymphocytes determined at the indicated time points ( n = 5). A representative staining of surface markers CD69+/CD25+ at day 2 from one subject is shown as scattergram. T2R38 expression (delta MESF) was assessed by Quantum Alexa Fluor 488 MESF beads in comparison to rabbit IgG isotype control; bars are means + SD (A,B) or means ± SD (C,D) . Significance of difference was calculated relatively to the respective control, * p < 0.05; ** p < 0.01. SC, solvent control. The gating strategy is shown in Supplementary Figure .

Article Snippet: The following primary human antibodies labeled with fluorophore were used for flow cytometry: CD3-APC (clone BW264/56), CD3-PE (clone REA613), CD4-PE (clone 15E8), CD4-APC (clone REA 623), CD8-PE (clone BW135/80), CD8-APC (clone BW135/80), CD45RO-Percp (clone UCHL1), CD62L-APC (Clone 145/15), CD69-PE (Clone FN50), CD11b (clone M1/70.15.11.5), CD14-PE (clone REA599) from Miltenyi Biotec (Bergisch Gladbach, Germany), CD19-APC (clone HIB19) from eBioscience, CD25-Percp (clone M-A251) from Bioledgen.

Techniques: Activation Assay, Expressing, Isolation, Staining, Comparison, Control, Solvent

Journal: Frontiers in Immunology

Article Title: Human T2R38 Bitter Taste Receptor Expression in Resting and Activated Lymphocytes

doi: 10.3389/fimmu.2018.02949

Figure Lengend Snippet: T2R38 expression status in T cell subsets. T2R38 expression was quantified in (A,B) freshly isolated cells ( n ≥ 6) or (C,D) CD3/CD28-stimulated cells after 72 h ( n ≥ 5). Each dot represents one donor, bars are means + SD . NA, naïve, CM, central memory, EM, effector memory cells. Representative histograms of different stains from one donor are shown. The gating strategy is given in Supplementary Figure . T2R38 expression (delta MESF) was assessed by Quantum Alexa Fluor 488 MESF beads in comparison to rabbit IgG isotype control. Significance of difference was calculated relatively to the respective control, * p < 0.05, ** p < 0.01.

Article Snippet: The following primary human antibodies labeled with fluorophore were used for flow cytometry: CD3-APC (clone BW264/56), CD3-PE (clone REA613), CD4-PE (clone 15E8), CD4-APC (clone REA 623), CD8-PE (clone BW135/80), CD8-APC (clone BW135/80), CD45RO-Percp (clone UCHL1), CD62L-APC (Clone 145/15), CD69-PE (Clone FN50), CD11b (clone M1/70.15.11.5), CD14-PE (clone REA599) from Miltenyi Biotec (Bergisch Gladbach, Germany), CD19-APC (clone HIB19) from eBioscience, CD25-Percp (clone M-A251) from Bioledgen.

Techniques: Expressing, Isolation, Comparison, Control