Anti cd3 pe cy7 clone sk7

PBMCs from CMV-positive donors were resuspended in RPMI media supplemented with 2% human AB serum (Corning, Celgro), and labeled with 5 μM CFSE (Molecular Probes, Life Technologies). CFSE-labeled PBMCs were inoculated with the recombinant strains of EBOV at an MOI of 2 PFU/cell, with or without simultaneous stimulation with 2 μg/ml of CMV pp65 peptides, SEB treated, or mock treated for 4 hours. PBMCs were washed twice to remove virus inoculum and cultured at a concentration of 2 x 10⁶ cells/ml for 7 days in Advanced RPMI medium supplemented with 10% human AB serum, 2 mM L-glutamine, 200 IU/ml penicillin, and 200 μg/ml streptomycin sulfate (Invitrogen) in 6 well plates. PBMCs were harvested, stained extracellularly with the following antibodies: anti-CD3 PE-Cy7 (clone SK7, BD Biosciences), anti-CD4-PerCP/Cy5.5 (clone SK3, BD Biosciences), anti-CD8 APC-Cy7 (clone SK1, BD Biosciences), and Live/Dead-Far Red Dead cell stain (Molecular Probes, Life Technologies). Cells were subsequently washed, fixed with 4% paraformaldehyde and removed from the BSL-4 according to an approved protocol. Cells were washed with PBS and analyzed by flow cytometry on BD LSR II.

Annexin-V-APC (0.1 μg/ml; BD Biosciences) was used for the assessment of phosphatidylserine exposure, propidium iodide (PI, 0.5 μg/ml) for cell viability analysis, and tetramethylrhodamine ethyl ester (TMRE, 20 nM) for mitochondrial transmembrane potential (ΔΨ_m) quantification. PCD was recorded in a FACSCanto II (BD Biosciences) in the total population (10,000 cells), and data were analyzed using FlowJo software. Chymotrypsin-like serine protease (serpase) or caspase cytofluorometric detection was performed with SerPase or Caspase activity kits from Imgenex (IMI-2301 and IMI-2315). Residual and leukemic B cells from CLL patients were discriminated from the mononuclear blood fraction by double labeling with anti-CD19 PerCP-Cyc5.5 (clone SJ25C1; BD Biosciences) and anti-CD5-PE-Cy7 mAb (clone L17F12; BD Biosciences). T cells from CLL patients were identified by a double anti-CD19 PerCP-Cyc5.5 and anti-CD3-PE-Cy7 (clone SK7; BD Biosciences) staining. Calreticulin cell surface exposure was recorded with anti-calreticulin-PE (clone FMC75; Assay Designs). CD47 analysis was assessed with conjugated anti-CD47-PE (clone B6H12; BD Biosciences), P21 with anti-P21-FITC (clone EA10; Calbiochem), and P53 with anti-P53-PE (clone DO-7; BD Biosciences). A QuantiBRITE flow cytometry system (BD Biosciences) was used to assess the number of CD47 molecules expressed on normal and CLL B lymphocytes.