Cd45ra apcalexafluor750

This was done as previously described (15 (link)). Briefly, 50 × 10³ target cells per well were placed in RPMI, 10% FBS, IL-2 100 U/mL with monensin (BD Biosciences) in a 96-well V-bottom plate. NK and target cells were incubated overnight at 37°C in 5% CO₂, and living cells were counted using a Muse cytometer (Millipore) with a count and viability kit (Millipore). As a control, NK cells were incubated without target cells. CD107a⁺ NK cells were analyzed on a Gallios flow cytometer (Beckman Coulter) using 7AAD, CD45RO-FITC, CD19-PE, CD56-PECy7, CD3-APC, CD45RA-APCAlexaFluor750, CD16-KromeOrange, and CD107a-HV500 (BD Biosciences). The results were analyzed using Kaluza software.

After PBMC purification and NK cell quantification, 3 million cells were incubated at 37°C for 4h or overnight with K562 target cells at an Effector (NK cell): Target ratio of 1:10 in a final volume of 500μl (RPMI Glutamax with 10% FBS and 10u/ml IL2). The medium also contained 1.5μl anti-CD107a antibody (BD Biosciences, Franklin Lakes, NJ) and 1μl monensin to prevent CD107a degradation (BD Golgi-Stop BD Biosciences). Then, cells were resuspended in 50μl of an antibody cocktail containing 7AAD, the anti-CD45RO-FITC, -CD69-PE, -CD19-ECD, -CD56-PECy7, -CD3-APC, -CD45RA-APCAlexaFluor750, -CD107a-HV500 and -CD16-KO antibodies (BD Biosciences, Beckman). Samples were analyzed on a Beckman Coulter FACS Gallios flow cytometer using the Kaluza software. Events were initially gated on forward and side scatter (SSC) to identify lymphocytes. A bivariate plot of CD56 versus CD3 was used to acquire at least 10,000 NK cells.