if possible, please verify method.identification column: filled in "antigen-loaded-targets,cultured-T-cells",
based on below methods section:
"Antigenic stimulation was performed on thawed PBMC. Individual HIV peptides were used at 10 μg/ml to stimulate HIV-specific CD8+ T cells for 14 h at 37°C in the presence of costimulation with anti-CD28 and anti-CD49d monoclonal antibodies (1-μg/ml [each] final concentration; BD Biosciences). In every experiment, a negative control (costimulation alone with anti-CD28/anti-CD49d) was included to account for spontaneous stimulation, and a positive control (Staphylococcus enterotoxin B [SEB], 1 μg/ml, final; Sigma-Aldrich) was included to ensure that the cells were responsive. After stimulation, the cells were surface stained with the following monoclonal antibodies: anti-CD3-fluorescein isothiocyanate (FITC), anti-CD8-Pacific Blue, anti-CD14-Cy5PE, anti-CD19-Cy5PE, anti-CD25-phycoerythrin (PE), and anti-CD69-allophycocyanin (APC) (all from BD Biosciences). Viable antigen-specific CD8+ T cells were identified on the basis of CD3, CD8, CD25, and CD69 coexpression as described previously for reactive T-cell populations (66). Staining for CD14 and CD19 was used to exclude macrophages and B cells from the analysis. Responding cell populations were sorted at 70 lb/in2 directly into 1.5-ml microtubes (Sarstedt) containing 100 μl RNAlater (Applied Biosystems) using a modified FACSAria flow cytometer (BD Biosciences); sort purity was >98% in all cases, and at least 1,000 cells were collected for each experimental condition. Unbiased clonotypic analysis of all expressed TRB gene products was then conducted using a nonnested template-switch-anchored reverse transcriptase PCR (RT-PCR) as described previously (28, 67). The entire procedure was performed blinded to the clinical status of the subjects in all cases. "
mikessh
commented
7 years ago
if possible, please verify method.identification column: filled in "antigen-loaded-targets,cultured-T-cells", based on below methods section:
"Antigenic stimulation was performed on thawed PBMC. Individual HIV peptides were used at 10 μg/ml to stimulate HIV-specific CD8+ T cells for 14 h at 37°C in the presence of costimulation with anti-CD28 and anti-CD49d monoclonal antibodies (1-μg/ml [each] final concentration; BD Biosciences). In every experiment, a negative control (costimulation alone with anti-CD28/anti-CD49d) was included to account for spontaneous stimulation, and a positive control (Staphylococcus enterotoxin B [SEB], 1 μg/ml, final; Sigma-Aldrich) was included to ensure that the cells were responsive. After stimulation, the cells were surface stained with the following monoclonal antibodies: anti-CD3-fluorescein isothiocyanate (FITC), anti-CD8-Pacific Blue, anti-CD14-Cy5PE, anti-CD19-Cy5PE, anti-CD25-phycoerythrin (PE), and anti-CD69-allophycocyanin (APC) (all from BD Biosciences). Viable antigen-specific CD8+ T cells were identified on the basis of CD3, CD8, CD25, and CD69 coexpression as described previously for reactive T-cell populations (66). Staining for CD14 and CD19 was used to exclude macrophages and B cells from the analysis. Responding cell populations were sorted at 70 lb/in2 directly into 1.5-ml microtubes (Sarstedt) containing 100 μl RNAlater (Applied Biosystems) using a modified FACSAria flow cytometer (BD Biosciences); sort purity was >98% in all cases, and at least 1,000 cells were collected for each experimental condition. Unbiased clonotypic analysis of all expressed TRB gene products was then conducted using a nonnested template-switch-anchored reverse transcriptase PCR (RT-PCR) as described previously (28, 67). The entire procedure was performed blinded to the clinical status of the subjects in all cases. "