Cd69 percp cy5 5 clone fn50

Thawed PBL which were allowed to rest overnight, as described above, were used for the in vitro bacterial stimulation assay. 150′000 cells/well of a 96 U-bottom well plate (TTP, Switzerland) were plated in duplicates in 270 μl of either control medium or medium containing inactivated bacteria (as described above). The plate was incubated at 37 °C and 5% CO₂ in humid atmosphere. Flow cytometry was used to identify activated T-lymphocytes at day four after plating. Analyzed were the early activation induced surface antigens CD69 and CD137. The measurement of CD69 and CD137 increases the sensitivity and optical discrimination of rare antigen specific T-cells [23 (link), 24 (link)]. PBL were stained with the following antibodies: CD4-FITC (clone RPA-T4, BD bioscience), CD8-APC (clone RPA-T8, BD bioscience), CD69-PerCP-Cy5–5 (clone FN50, BD bioscience) and CD137-PE (clone 4B4–1, BioLegend). Cells were incubated with antibodies for 15 min at room temperature, washed and resuspended in PBS. Cell fluorescence was evaluated with FACScalibur flow cytometer and data were analyzed using FlowJo, 10.0 software. Activated cells were characterized as being CD69⁺CD137⁺ and the percentage of bacteria-specific T-cells was calculated by subtracting percentage of the activated unstimulated T-cells from the percentage of the activated bacterial stimulated T-cells.

Fixable Viability Stain 510 was obtained from ThermoFisher Scientific (#L34957, 1/1000). The following antibodies were used in staining experiments: CD3 AF700 Clone UCHT1 (BD Biosciences, #557943, 1/50), CD4 PE-Cy7 Clone L200 (BD Bioscience, #560644, 1/200) and CD4 BV786 Clone SK3 (BD Biosciences, #563881, 1/200), CD8 BV510 Clone RPA-T8 (BioLegend, #301047, 1/200), CD45RO BV421 Clone UCHL1 (BD Biosciences, #562649, 1/100) or CD45RO PE Clone HI100 (BD Biosciences, #5555493, 1/20), CD27 BV605 Clone L128 (BD Biosciences, #562656, 1/100), PD1 BB700 Clone EH12.1 (BD Biosciences, #566461, 1/100), CD69 PerCP-Cy5.5 Clone FN50 (BD Biosciences, #560738) and CD69 APC (BioLegend, #310909, 1/50), CD25 BV421 Clone M-A251 (BD Biosciences, #562443, 1/50), HLA-DR FITC Clone REA805 (Miltenyi Biotech, #130-111-788, 1/50), GZMA PE-Cy7 Clone CB9 (BioLegend, #507221, 1/25), GZMB PB Clone GB11 (BioLegend, #515407, 1/50), CCL5 PerCP-Cy5.5 Clone VL1 (BioLegend, #515507, 1/50), CD127 PE-CF594 Clone HIL-7R-M21 (BD Biosciences, #562397, 1/50). For p24 staining, we used a combination of two antibodies: p24 KC57-FITC (Beckman Coulter, #6604665, 1/500) and p24 28B7-APC (MediMabs, #MM-0289-APC, 1/400).