Journal:

Article Title: Anti-TNF immunotherapy reduces CD8 + T cell-mediated antimicrobial activity against Mycobacterium tuberculosis in humans

doi: 10.1172/JCI38482

Figure Lengend Snippet: PBMCs from healthy donors were stained with PE-Cy5-5–conjugated anti-CD4, PE-conjugated anti-CD56, allophycocyanin-conjugated anti-CD19, PerCP-conjugated anti-CD8 (filled histograms), or appropriate isotype controls (open histograms). All samples were then stained for granulysin using FITC-conjugated donkey anti-rabbit as a secondary antibody. Granulysin+ cells were gated according to the isotype (control rabbit serum, not shown) and analyzed for the expression of cell surface markers. The percentages of CD4+, CD56+, CD19+, and CD8+ cells within the granulysin gate are indicated. Shown is a typical result of 27 donors.

Article Snippet: The following antibodies were used for flow cytometry: PE-Cy5.5–conjugated anti-CD4, FITC-conjugated anti-CD16, allophycocyanin-conjugated anti-CD19, allophycocyanin-conjugated anti-CD45RA, biotin-conjugated anti-mouse IgG1, and allophycocyanin/PE-conjugated streptavidin (all from Caltag); PerCP/allophycocyanin-conjugated anti-CD8, PE/allophycocyanin-conjugated anti-CD56, and PE-conjugated anti-perforin (all from BD Biosciences); FITC-conjugated anti-CCR7 (R&D Systems); FITC-conjugated anti-Vα24 (Immunotech); PE-conjugated anti-CD161 (Beckman Coulter); polyclonal granulysin rabbit serum (gift from A. Krensky, Stanford University, Stanford, California, USA); infliximab (Essex Pharma); etanercept (Wyeth Pharma); and Cy5-conjugated donkey anti-rabbit and biotin-conjugated goat anti-rabbit (Dianova).

Techniques: Staining, Expressing

Journal:

Article Title: Anti-TNF immunotherapy reduces CD8 + T cell-mediated antimicrobial activity against Mycobacterium tuberculosis in humans

doi: 10.1172/JCI38482

Figure Lengend Snippet: PBMCs from healthy donors were stained with PerCP- or allophycocyanin-conjugated anti-CD8 and anti-granulysin (detected with donkey anti-rabbit biotin and streptavidin) or PE-conjugated perforin. Additional labeling was performed using allophycocyanin-conjugated anti-CD45RA and FITC-conjugated anti-CCR7 to detect TEM cells, PE-conjugated anti-CD161 and FITC-conjugated anti-Vα24 to detect NKT cells, and PE-conjugated CD56 and FITC-conjugated anti-CD16 to detect NK cells. Samples with appropriate isotypes were included in all experiments. (A) CD8+granulysin+ cells were gated, and the expression of additional markers within this gate was determined. Numbers denote the percentage of positive effector cells, NKT cells, or NK cells within the population of CD8+granulysin+ cells. For each sample, 1 × 106 cells were acquired. Shown is a typical result of 8 donors. (B) CD8+perforin+ cells were gated, and the expression of additional markers within this gate was determined. Numbers denote the percentage of positive effector cells, NKT cells, or NK cells within the population of CD8+perforin+ cells. For each sample, 1 × 106 cells were acquired. Shown is a typical result of 4 donors.

Article Snippet: The following antibodies were used for flow cytometry: PE-Cy5.5–conjugated anti-CD4, FITC-conjugated anti-CD16, allophycocyanin-conjugated anti-CD19, allophycocyanin-conjugated anti-CD45RA, biotin-conjugated anti-mouse IgG1, and allophycocyanin/PE-conjugated streptavidin (all from Caltag); PerCP/allophycocyanin-conjugated anti-CD8, PE/allophycocyanin-conjugated anti-CD56, and PE-conjugated anti-perforin (all from BD Biosciences); FITC-conjugated anti-CCR7 (R&D Systems); FITC-conjugated anti-Vα24 (Immunotech); PE-conjugated anti-CD161 (Beckman Coulter); polyclonal granulysin rabbit serum (gift from A. Krensky, Stanford University, Stanford, California, USA); infliximab (Essex Pharma); etanercept (Wyeth Pharma); and Cy5-conjugated donkey anti-rabbit and biotin-conjugated goat anti-rabbit (Dianova).

Techniques: Staining, Labeling, Expressing