Cd4 fluorescein isothiocyanate fitc

The frequencies of PD-1 and PD-L1 expressions were assessed using a Beckman Coulter flow cytometer (FC500; Beckman Coulter, Inc., Brea, CA, USA) in all studied individuals according to the stain-lyse and wash protocol as previously described by Moniuszko et al (19 (link)). Briefly, 50 µl EDTA-anti-coagulated whole blood was stained with 5 µl of the following mouse anti-human monoclonal antibodies, CD4 fluorescein isothiocyanate (FITC; cat no. 317408), CD8a-FITC (cat no. 301006), CD14-FITC (cat no. 325604), PD-1-phycoerythrin (PE; cat no. 329906) and PD-L1-PE (cat no. 329706) to identify PD-1⁺CD4⁺, PD-1⁺CD8⁺ T cells and CD14⁺PD-L1⁺ monocytes, respectively (all used as supplied and obtained from BioLegend, Inc., San Diego, CA, USA). All blood samples were incubated for 30 min at 4°C in the dark. Thereafter, the cells were lysed using Optilyse C Lysis Solution (Immotech SAS, Marseille, MC, France) and washed twice with cold PBS. A minimum of 100,000 events was acquired. FlowJo version 7.6.1 (Tree Star, Inc., Ashland, OR, USA) software was used for the analysis of flow cytometry data.

At day 7 postprime, mouse splenocytes were collected and seeded to the plate (1 × 10⁶ per well). The RBD peptide pool or influenza A HA peptide pool was added to stimulate splenocytes for 12 hours at 37°C, respectively. Brefeldin A solution was added into the plate with a final concentration of 1 μg/ml and incubated for another 4 hours. Following that, the splenocytes were collected and stained with CD4–fluorescein isothiocyanate (FITC) (100406, BioLegend) antibody or CD8-FITC (ab22504, Abcam) antibody at a dilution of 1:100. After washing with magnetic-activated cell sorting flow buffer, splenocytes were fixed with 4% paraformaldehyde (PFA) and permeabilized using saponin and incubated with IFN-γ–phycoerythrin (PE) (507806, BioLegend), IL-4–PE (504103, BioLegend), or IL-17a–APC (17-7177-81, Invitrogen) Abs for 1 hour. The stained splenocytes were analyzed by a CytoFLEX flow cytometer (Beckman Coulter). Data analyses were carried out by FCS Express V6.