8 color navios flow cytometer

Fresh PB or SF samples were processed within a few hours after sample collection. One hundred microlitres of whole PB in EDTA, or 5 × 10⁵ mononuclear cells derived from SF (separated by Ficoll-Hipaque (Cederlane, Ontario, Canada) density gradient centrifugation) in 100 μl of phosphate-buffered saline were first incubated in the dark at RT for 20 min with anti-human antibodies specific for CD45 (APC-A750) (clone J33) (dilution 1/25), CD19 (APC-700) (clone J3-119) (dilution 1/25), CD38 (PC5 or APC) (clone LS198-4-3) (dilution 1/25), CD27 (PC7) (clone 1A4CD27) (dilution 1/25) and FITC-conjugated IgD (clone IA6-2) (dilution 1/25) (all by Beckman Coulter, Marseille France). After staining, the cells were fixed, washed and erythrocytes were lysed. Samples were immediately analysed on optimally compensated 8 color Navios flow-cytometer and data were analysed with Kaluza software (Beckman Coulter, Marseille, France). Lymphocytes were gated on the basis of forward and side-scatter light properties (confirmed by CD45 staining) and at least 10,000 CD19⁺ cells were analysed. B-cell subsets were evaluated by the expression of surface B-cell markers according to IgD/CD27 classification47 (link).

Samples of venous blood (30 mL) were drawn into ethylene diamine tetra acetic acid (EDTA) tubes, carried by hand to the laboratory, and immediately processed for flow cytometer evaluation. To block unspecific binding of antibodies, cells were incubated with 2.5 μg/mL mouse IgG (Sigma-Aldrich, St. Louis, MO, USA, Cat-no: I8765-10MG) diluted in phosphate buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA, Ref-no: 14190-094) for 15 min on ice. Next, the cells were stained with fluorochrome-labeled monoclonal antibodies.
The following anti-human antibodies for staining of cell surface markers were used: CD3-ECD (clone UCHT1), CD4-FITC or -PECy7 (clone SFCI12TAD11), CD8-FITC (clone B9.11), CD56-APC (clone N901), TCR PAN αβ-PE (clone IP26A), TCR PAN γδ-FITC (clone IMMU 510), CD45-PECy5.5 or -ECD (clone J.33), CD20-PECy5.5 (clone B9E9) were purchased from Beckman Coulter Inc. Brea, CA, USA. Data were acquired by an 8-color NAVIOS^® flow cytometer (Beckman Coulter Inc., Brea, CA, USA) and analyzed using Kaluza Analysis Software (Beckman Coulter Inc. Brea, USA.). We acquired 100,000 for each sample.