Cd8a alexa fluor700

Spleens are isolated from mice at 1 week after the last injections. Fresh splenocytes are divided into two parts. One part stimulated with RBD/HIV Env epitope pool (5 μg/mL) for 4 − 6 h at 37 °C and 5% CO₂. The viability of splenocytes was assessed using Zombie NIRIM Fixable Viability Kit, washed by cell staining buffer and blocked with anti-FcR antibodies, and stained with surface antibodies as following: CD3-Billiant Violet 510, CD4-FITC, CD8a-Alexa Fluor 700 (Biolegend, the United States). After 30 min incubation at 4 ℃, splenocytes were washed twice by cell staining buffer, fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Kit. Splenocytes were stained 30 min at 4 ℃ with intracellular antibodies of IFN-γ-APC. The viability of the other splenocytes was assessed using Zombie NIRIM Fixable Viability Kit, washed by PBS and blocked with anti-FcR antibodies, and stained with surface antibodies as following: CD45R-B220, H-2kd-FITC, CD11c-BV605, CD80-PE and CD86-PE/cy7 (Biolegend, the United States). After completing the above staining steps, cells were washed twice by PBS with centrifugation. Cells were resuspended in 200 μL cell staining buffer. All samples were acquired on a BD FACS Fortessa and results were analyzed with the FlowJo software v10.7.1.4.