Anti cd69 pe cy5

MALC obtained at day 10 of growth, were co-cultured with NK cells at ratio E:T 0.5:1 or 1:1 and treated or not with mAbs at 10 μl/ml during 4 h in presence of brefeldin A at 10 μl/mL (except of CD69 detection). Cells were then washed, fixed with 2% PFA during 4 min, washed and permeabilized with 1% saponin during 8 min. Cells were stained with anti-CD56-PE (Miltenyi Biotec), anti-CD3-PE/Cy7 (Beckman Coulter, Roissy, France), anti-CD69-PE-Cy5 (BD Biosciences), anti-perforin (PFN)-FITC (BD Biosciences), anti-IFN-γ-Alexa fluor 647 (BD Biosciences), anti-granzyme B-Alexa647 (BD Biosciences) antibodies during 30 min at 4°C in the dark. Cells were analyzed by flow cytometry with a LSRII flow cytometer (BD Biosciences).

Fifty microliters of whole blood was mixed with the following fluorochrome-conjugated antibodies: anti-CD3/FITC (IM2635 Immunotech), anti-CD19/PerCP (MHCD1931/Invitrogen), anti-CD14/PE-Cy7 (557742/BD), anti-CD62L/APC (MHCD62L05/Invitrogen), anti-CD69/PECy5 (555532BD) and anti-CD86/PE (MHCD8604 Invitrogen). Appropriate isotype controls for each antibody were used. After 15 min in the dark at room temperature (RT), 500 µL of working FACS Lysing solution (Becton-Dickinson, CA, USA) was added, and the reaction was incubated for 10 min at RT. The cell suspensions were washed once with a 1-mL fraction of phosphate-buffered saline by centrifugation at 900×g for 5 min at RT and then analyzed. Ten thousand total single events were acquired in a FACS Aria I flow cytometer (BD, biosciences USA) equipped with the FACSDiva 6.1.3 software (BD PharMingen). The analysis was performed using the Infinicyt Analytical Software (Cytognos). The percentages of CD62L, CD69 and CD86 expressing cells were determined from the CD3, CD19 or CD14-gated positive events from the SSC vs CD3, CD19 or CD14 respective dot plots.