Cd5 ecd

B cell whole peripheral blood samples (200 μL) were stained with the following anti-human monoclonal antibodies IgD-Alexa Fluor 488 (cat. 348216, BioLegend, Inc., San Diego, CA, USA), CD38-PE (cat. A07779, Beckman Coulter, Brea, CA, USA), CD5-ECD (cat. A33096, Beckman Coulter, USA), CD27-PC7 (cat. A54823, Beckman Coulter, Brea, CA, USA), CD19-APC/Cy7 (cat. 302218, BioLegend, Inc., USA), and CD45-Krome Orange (cat. A96416, Beckman Coulter, USA), all antibodies were utilized at the dilutions that were recommended by the manufacturers. After incubation at room temperature in the dark for 10 min, erythrocytes were lysed for 15 min by adding 2 mL of VersaLyse Lysing Solution (Beckman Coulter, Inc., USA) supplied with 50 μL IOTest 3 Fixative Solution (Beckman Coulter, Inc., USA). Next, cells were washed (7 min, 330 g) twice with a buffer (sterile phosphate-buffered saline (PBS) containing 2% of heat inactivated fetal bovine serum, Sigma-Aldrich, St. Louis, MO, USA) and were resuspended in 0.5 mL PBS containing 2% of neutral buffered formalin solution (Sigma-Aldrich, St. Louis, MO, USA). Sample acquisition was performed using a Navios flow cytometer (Beckman Coulter, Inc., USA), equipped with 405, 488 and 638 nm lasers. There were collected at least 5000 CD19+ B cells to be analyzed in each sample.

Antibodies directed against the following human cell surface proteins were obtained from commercial sources: CD45-PC7, CD45-FITC, CD3-PE, CD5-ECD, CD34-FITC, CD19-APC, CD23-PE (Beckman Coulter, Miami, FL) CD133-PE and CD133-APC (Miltenyi Biotec, Auburn, CA). Stained samples were washed in PBS containing 2% FBS and 7-aminoactinomycin (7-AAD). A minimum of 20,000 cells (unless noted otherwise) was analyzed on a Coulter Cytomics FC500 flow cytometer (Beckman Coulter, Miami, FL). All analysis of normal and lymphoma cells were gated on human CD45+ cells. Sequential gates were set to include only viable cells and quadrant markers were set to exclude at least 99.9% of cells labeled with the appropriate fluorochrome labeled isotype controls.

B-cell whole peripheral blood samples (200 μL) were stained with the following anti-human monoclonal antibodies: IgD-Alexa Fluor 488 (cat. 348216, BioLegend, Inc., San Diego, CA, USA), CD38-PE (cat. A07779, Beckman Coulter, Brea, CA, USA), CD5-ECD (cat. A33096, Beckman Coulter, Brea, CA, USA), CD27-PC7 (cat. A54823, Beckman Coulter, Brea, CA, USA), CD19-APC/Cy7 (cat. 302218, BioLegend Inc., San Diego, CA, USA), and CD45-Krome Orange (cat. A96416, Beckman Coulter, Brea, CA, USA). All antibodies were utilized at the dilutions that were recommended by the manufacturers. After incubation at room temperature in the dark for 15 min, red blood cells were lysed for 15 min by adding 2 mL of VersaLyse Lysing Solution (Beckman Coulter, Inc., Brea, CA, USA) supplied with 50 μL IOTest 3 Fixative Solution (Beckman Coulter, Inc., Brea, CA, USA). Next, cells were washed (7 min, 330 g) twice with a buffer (sterile phosphate-buffered saline (PBS) containing 2% of heat-inactivated fetal bovine serum, Sigma-Aldrich, St. Louis, MO, USA) and were resuspended in 0.5 mL of PBS containing 2% of neutral buffered formalin solution (Sigma-Aldrich, St. Louis, MO, USA). Sample acquisition was performed using a Navios flow cytometer (Beckman Coulter, Inc., Brea, CA, USA), equipped with 405, 488, and 638 nm lasers. At least 5000 CD19+ B-cells were collected for analysis from each sample.