Anti cd95 apc

EDTA anticoagulated peripheral blood was used for phenotype analysis by flow cytometry. In detail, 200 μL of whole blood was lysed in 2 mL of VersaLyse at room temperature for 15 min. Then, cells were washed twice with FACS buffer, followed by resuspension in the appropriate antibody preparation and incubated for 20 min at 4°C. For intracellular staining of Ki-67 expression, we used permeabilization and fixation method using eBioscience perm/fix kit (ebioscience cat no. 88-8824-00). The following monoclonal antihuman antibodies were used in appropriate concentrations to stain cells: anti-CD45-Krome orange (Beckman Coulter, cat no. 96416), anti-CD14-PC5.5 (Beckman Coulter, cat no. A70204), anti-CD 19-APC-Alexa fluor 750 (Beckman Coulter, cat no.A94681), anti-CD27-PE (BD Pharmingen, cat no.555441), anti-CXCR3-APC (BD Pharmingen, cat no. 561732), anti-CXCR4-APC (BD Pharmingen, cat no. 560936), anti-CD95-APC (BD Pharmingen, cat no. 558814), anti-ki-67-PE (Ebioscience Cat no. 12-5699-42), and anti- IgD-FITC (BD Pharmingen, cat no. 555778). After staining, the cells were analyzed by 10-color flow cytometry (Navios, Beckman Coulter), with at least 20,000 CD19+ events collected for each analysis.

The cells were trypsinized and washed twice in PBS (PanEco, Moscow, Russia) supplemented with 1% FBS (Gibco, Waltham, MA USA) by centrifugation for 5 min at 300× g. The cells were incubated with anti-CD14-FITC, anti-CD34-APC, anti-CD45-PE, anti-CD29-APC, anti-CD44-FITC, anti-CD73-PE, anti-CD105-PE, anti-CD95-APC (BD Biosciences, Franklin Lakes, NJ, USA) at concentrations recommended by the manufacturer, for 1 h at 4 °C in the dark. At the end of the incubation, the cells were washed twice in PBS by centrifugation. Fluorescence intensity was analyzed using BD FACSAria III flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). At least 10⁴ events were recorded in each sample. The results were processed using the FlowJo_V10 (FlowJo™, Ashland, OR, USA). The relative fluorescence intensity (RFI) of marker expression was calculated as the ratio of the specific fluorescence of cell staining with fluorescently labeled antibodies and the autofluorescence of control unstained cells.

T cell subpopulations in peripheral blood were analyzed by flow cytometry. The following conjugated antibodies were purchased from BD Biosciences (San Diego, CA, USA): anti-CD4-FITC, anti-CD4-PE-Cy7 and anti-CD4-PerCP (L200); anti-CD8-APC-Cy7 (RPA-T8); anti-CD45-PE (DO58-1283); anti-CD3-PerCP and anti-CD3-Pacific Blue (SP34-2); anti-CD28-FITC (CD28.2); anti-CD95-APC and anti-CD95-PE cy5 (DX2); and anti-CCR5-PE (3A9). The conjugated antibodies anti-CD8-APC and anti-CD8-PE (B9.1) were obtained from Immunotech SAS (Marseille Cedex, France). The number of lymphocytes in peripheral blood samples was determined using the BD TruCOUNT kit (BD Biosciences, San Jose, CA, USA). Cell surface marker staining for flow cytometry was performed as follows: 50 μl of EDTA-treated whole blood was incubated with antibodies at room temperature (RT) for 20 min in the dark, and red blood cells were lysed in Lysing Buffer (BD Pharmingen) at RT for 10 min. Following a wash with PBS containing 2% FBS, the cells were analyzed by flow cytometry on either a FACSCalibur (BD Biosciences) or FACSAria (BD Biosciences) system. The data were analyzed using FlowJo software (Tree Star, Inc., USA).