Extracellular and intracellular labelling for flow cytometry analysis

Cryopreserved PBMCs (0.5–106) from patients and controls were thawed in water bath at 37 °C, centrifuged (350 g, 5 min) and washed once with 1 mL of PBS pH 7.4 supplemented with 2% FCS and 0,01% NaN3. Thawed cells were stained with panels comprising the antibodies listed above for flow cytometry analysis. Cells were labeled extracellularly and intracellularly as described previously¹³. Briefly, after extracellular stain with cocktail surface markers, cells were washed in Perm/Wash for 15 min and then were incubated with Cytofix/Cytoperm solution for 20 min at 4 °C (BD biosciences). Cells were washed again in Perm/Wash and stained for 30 min with PE conjugated anti apoptotic Bcl-2 protein mAb. About 1 × 10⁵ events were acquired in the lymphocyte and monocyte gates. Data acquisition and analysis were performed using FACS Aria IIu and analyzed using FlowJo software version 10 respectively.