Ecd anti cd19 clone 6d5

The spleen was dissociated into RPMI 1640 medium. Red blood cells were lysed with NH₄Cl 140 mM (eBioscience, San Diego, CA, USA) before counting nucleated cells using a hemocytometer. To evaluate lymphocyte sub-populations, 10⁶ splenic cells were incubated for 15 min at 4°C in the dark with a mixture of four anti-mouse fluorescence-labeled monoclonal antibodies: ECD anti-CD19 (clone 6D5, Beckman Coulter, Marseille, France), APC anti-CD3ε (clone 17A2, eBioscience), PE anti-CD4 (clone RM4-5, eBioscience) and PE-Cy7 anti-CD8α (clone 53-6.7, eBioscience). Immunophenotyping was carried out using a five-color FC500 flow cytometer (Beckman Coulter). A first gate was applied to select the lymphocyte population using a window with forward scatter (FSC) and side scatter (SSC). Fifty thousand events in the viable lymphocyte gate were acquired. Data were analyzed using FlowJo software v7.6.5 (Tree Star Inc., Ashland, OR, USA) to identify specific lymphocyte sub-populations: CD19⁺ B cells, CD3⁺ T cells, CD3⁺CD8⁺ cytotoxic T cells (Tc) and CD3⁺CD4⁺ helper cells T (Th). Populations are expressed as percentages of the total lymphocyte population.

Spleens were dissociated in RPMI 1640 medium. Red blood cells were lysed with NH₄Cl 140 mM (eBioscience, San Diego, CA, United States) before counting nucleated cells with a hemocytometer. To evaluate lymphocyte sub-populations, 10⁶ splenocytes were incubated for 15 min at 4°C in the dark with a mixture of four anti-mouse monoclonal antibodies: ECD anti-CD19 (clone 6D5, Beckman Coulter, Marseille, France), APC anti-CD3𝜀 (clone 17A2, eBioscience), PE anti-CD4 (clone RM4-5, eBioscience) and PE-Cy7 anti-CD8α (clone 53-6.7, eBioscience). Immunophenotyping was carried out using a five-color FC500 flow cytometer (Beckman Coulter). Data were analyzed using the FlowJo v7.6.5 software (Tree Star Inc., Ashland, OR, United States).