Anti cd5 pe cy7

Blood samples and bone marrow aspirate for immunophenotype analysis was obtained prior to plerixafor administration on day -5 and after its administration on day -4 (prior to thiotepa administration). The cell content was phenotyped by flow cytometry using BD FACSCanto™ II flow cytometer, BD FACSDiva 6.0 software, and a red cell lysis/multi-color antibody protocol. The following monoclonal antibodies against cell surface or intracellular markers were used: Anti-CD45 APC-H7 (Clone 2D1), Anti-CD33 PE-Cy7 (Clone P67.6), Anti-sCD3 V450 (Clone UCHT1), Anti-CD7 FITC (Clone M-T701), Anti-CD5 PE-Cy7 (Clone L17F12), Anti-CD19 APC (Clone SJ25C1), Anti-CD33 APC (Clone P67.6), Anti-HLADR APC-H7 (Clone L243), Anti-CD184 (CXCR4) PE (Clone ID9), Anti-IgG 2a PE (Clone X-39; all 6from BD Biosciences, San Jose, CA); Anti-CD34 PerCP (Clone 581), Anti-cCD3 PerCP (Clone SK7; all from Biolegend, San Diego, CA); Anti-CD38 FITC (Clone T16; Beckman Coulter, Pasadena, CA) and Anti-CD133 APC (Clone AC133, Miltenyi Biotec, Cambridge, MA). Patient-specific combinations of 6 or 8 antibodies and Boolean gating scheme were used to identify the blasts for each patient and determine their CXCR4 expression. Matched isotype control was used to determine the upper limit of fluorescent background.

PBMCs from CLL samples were stained with anti-CD19 APC, anti-CD5 PE-Cy7, and anti-CD3 APC-H7 antibodies (BD Biosciences) to identify CLL cells. Cells were pre-incubated with Aqua Blue dead cell exclusion dye (Invitrogen), followed by surface staining using anti-CD49d PE antibody and acquired on LSRFortessa (BD Biosciences) using FMO and isotype controls. Data were analyzed using FlowJo (Version 10; TreeStar), and were reported as a percentage of positive cells within the CLL population.