Rat igg2a k isotype control pe

Isolated peripheral blood leukocytes were analyzed by triple immunofluorescence staining, followed by flow cytometry. The following primary antibodies were purchased from eBiosciences (San Diego, CA, USA): anti-allophycocyanin (APC)-labeled CD3 (clone 145-2C11), anti-fluorescein isothiocyanate (FITC)-labeled CD4 (clone GK 1.5), anti-phycoerythrin (PE)-labeled CD8 (clone 53–6.7), Armenian Hamster IgG Isotype Control APC (clone eBio299Arm), Rat IgG2b K Isotype Control FITC (eB149/10H5), and Rat IgG2a K Isotype Control PE (eBR2a). The cells were stained with a mixture of the primary antibodies at 4°C for 30 min. Flow cytometry was performed on a fluorescence- activated cell sorting (FACS) Calibur instrument (BD Biosciences, San Jose, CA, USA).

Blood samples were collected into sterile tubes containing EDTA anticoagulant. Fifty microliters of blood was incubated (in the absence of light) in TruCount^TM tubes with lymphocyte subset antibodies (anti-mouse CD3 APC-eFluor 780 17A2, anti-mouse CD4 APC GK1.5, anti-mouse CD8a PE 53-6.7, and anti-mouse CD19 PE-Cy7 eBio1D3 (1D3) were from eBioscience, USA ) at 25 °C for 15 min. Matched labeled isotype antibodies were used as negative controls (rat IgG2b K Isotype control APC-eFluor 780, rat IgG2b K Isotype control APC, rat IgG2a K Isotype control PE, and rat IgG2a K Isotype control PE-Cy7 were from eBioscience, USA). Then, the samples were treated with 450 μL 1× BD FACS lysing solution (BD Company, USA). After the erythrocytes were lysed, 10,000 cells along with beads were acquired on a FACSCanto™ II flow cytometer (BD Company, USA). The results were analyzed using FACSDiva software (BD Company, USA).