Cd3 apc cy7 sk7

Flow cytometric assays were performed as described previously (11 (link), 12 (link)). Briefly, PBMCs were thawed and co-cultured in a 24-well plate at 1.0 × 10⁶ cells/well in RPMI 1,640 with 10% fetal calf serum for 24 h at 37°C and 5% CO₂. After overnight culture, the cells were labeled with the following anti-human monoclonal antibodies: CD3-APC Cy7 (SK7; BD Bioscience), CD4-Alexa700 (RPA-T4; BD Bioscience), CD8-BV480 (PRA-T8, BD Bioscience), CD19-BV421 (HIB19, Bio-Legend), HLA-DR-BV605 (G46-6, BD Bioscience), CD38-BV786 (HIT2, BD Bioscience), PD1-phycoerythrin (PE) (EH12.1, Bio-Legend), CD25-PECy7 (BC96; Bio-Legend), and IL7Ra (CD127)-BV650 (A019D5; Bio-Legend). Isotype controls, 7-AAD, and Fc blocker were included in all experiments. Multicolor flow cytometric analysis was performed using a BD FACS Fortessa analyzer (BD Biosciences) and FlowJo ver. 10 software (Tree Star Inc., Ashland, OR).

Whole fresh blood samples were prepared immediately after collection for immunophenotyping. Immune cell populations were characterized by surface staining with the following fluorescence-labeled antibodies according to the manufacturers´ instructions: HLADR-BV510 (G46-6; BD Bioscience), CD3 APC Cy7 (SK7, BD Bioscience), CD45-FITC (HI30, BD Bioscience), CD14-PECF 594 (MφP9, BD Bioscience), CD19-PE Cy5 (HIB 19, BD Bioscience), CD8-PerCP CY5.5 (RPA-T8, BD Bioscience), CD4-PE Cy7 (RPA-T4, BD Bioscience), and CD56-BV785 (5.1H11, Biolegend). For the lysis of red blood cells, FACS lysing solution (BD Bioscience) was added. Viability dye (VD, eBioscience) staining was used to evaluate apoptotic cells. Cell frequencies were evaluated on the LSR Fortessa cytometer (BD Bioscience, San Jose, CA, USA). Gating strategy for cells is presented in Supplementary Figure S1.

Coulter (BC; Hialeah, FL) or BD was utilized for the fluorescently labeled antibodies. Antibodies used in the single six-color FC tube for all the specimens (antibody clone designation and supplier) included CD64-FITC (22; BC), CD30-PE (Ber- H83; BD), CD40-PE-Cy5.5 (MAB89; BC), CD20-PE-Cy7 (B9E9; BC), CD95-APC (DX2; BD), and CD3-APC-Cy7 (SK7; BD) or CD3-APC-H7 (SK7; BD). To determine background fluorescence, an appropriate fluorescence minus one control was also utilized. Extensive lymphoma panel was used for FC immunophenotyping which included the following antibodies: CD8-fluorescein isothiocyanate (FITC)/CD4-phycoerythrin (PE)/CD3-Texas Red (ECD)/CD7-PE cyanine 5 (PC5), FITC/CD19-PE/CD10-ECD/CD5- PC5, FITC/CD19-PE/CD10-ECD/CD5-PC5,FMC7-FITC/CD103-PE/CD20-ECD/CD19-PC5,CD38-FITC/CD23-PE/CD19-ECD/CD138-PC5,andCD16+CD56-FITC/CD25-PE/ CD3-ECD/CD14-PC5; all tubes contained CD45-PE cyanine 7 (PC7).The panel was altered, if necessary, based on clinical data, limited material, concurrent morphologic assessment, or previously known lymphoma subtype. Clinical follow-up of greater than six months was required for a negative flow cytometric study without a subsequent biopsy. The data analysis was done using Statistical Package for the Social Sciences (SPSS) version 21. One-way ANOVA was used to compare diagnosis with a number of antibodies used.