Zenon alexa fluor 488 mouse igg2a labeling kit

Sample of PBMC cells were stained with the MoAbs anti-CD19 PE-Cy7, CD5 APC or CD3 PE (BD Pharmingen). After membrane staining, the cells were fixed by 1% paraformaldehyde solution. Next, anti-ZAP-70 antibody (Biomol Research Laboratories, USA) that was labeled by the ZenonTM Alexa Fluor^® 488 Mouse IgG2a Labeling Kit (Molecular Probes, USA) was added to the sample tubes. The samples were incubated for 30 min, washed, and examined by flow cytometry method. When ZAP-70 expression was detected in ≥ 20% of leukemic cells, the subject was considered positive for ZAP-70. To assess CD38 expression, PBMC were stained with anti-CD38 FITC, anti-CD19 PE, anti-CD5 CyChrome MoAbs, or IgG1 isotypic control (BD Pharmingen) for flow cytometry analysis. Patients were considered CD38 positive when expression was found in at least 20% of CLL cells.

A total of 1 × 10⁶ peripheral blood cells were stained with the monoclonal antibodies CD19 PE (BD Pharmingen), CD5 CyChrome (Caltag Laboratories, USA), or CD3 PE (BD Pharmingen). Following membrane staining, the cells were fixed in 1 % paraformaldehyde solution in PBS for 15 min at room temperature and permeabilized with 70 % ethanol for one hour at −20 °C. After washing with PBS, anti-ZAP-70 antibody (Biomol Research Laboratories, USA) labelled by the ZenonTM Alexa Fluor® 488 Mouse IgG2a Labeling Kit (Molecular Probes, USA) was added to the sample tubes. The samples were incubated with the reagent for 30 min at room temperature, washed once with PBS, and analyzed by flow cytometry (FACSCalibur, Becton Dickinson).
To assess CD38 expression, peripheral blood mononuclear cells were stained with CD38 FITC, CD19 PE, CD5 CyChrome, or IgG1 isotypic control MoAbs. The cells were incubated for 20 min at room temperature. Finally, the cells were washed and analyzed by flow cytometry.

A total of approximately 1 × 10⁶ peripheral blood cells were stained with the mAbs CD19 PE-Cy7, CD5 APC (Pharmingen, USA) or CD3 PE (BD Pharmingen). Following membrane staining, the cells were fixed by 1 % paraformaldehyde solution in phosphate-buffered saline for 15 min at room temperature and permeabilized with 70 % ethanol for 1 h at −20 °C. After washing, anti-ZAP-70 antibody (Biomol Research Laboratories, USA) that was labeled by the ZenonTM Alexa Fluor® 488 Mouse IgG2a Labeling Kit (Molecular Probes, USA) was added to the sample tubes. The samples were incubated for 30 min, washed, and examined by flow cytometry method. Patients were considered positive for ZAP-70 when its expression was detected in ≥20 % of leukemic cells. To assess CD38 expression, peripheral blood mononuclear cells were stained with anti-CD38 FITC (BD Pharmingen), anti-CD19 PE, anti-CD5 CyChrome mAbs, or IgG1 isotypic control for 20 min in darkness, and they were analyzed with flow cytometry method. Patients were considered CD38 positive when expression was found in at least 20 % of CLL cells.