Apc labeled anti mouse cd4 antibody

Splenocytes from immunized mice (1x10⁶/200 μL) were incubated in the presence or absence of rTc80 (10 μg/ml) with complete RPMI medium in 96-well U-bottom plates. After 4 h of incubation, brefeldin A was added at 10 μg/ml and 12 h later, surface and intracellular cytokine staining was carried out.
Splenocytes were incubated with an APC-labeled anti-mouse CD4 antibody (eBioscience Inc.) for 45 min at 4°C, fixed with 2% PFA for 15 min at RT and incubated for 45 min at 4°C in permeabilization buffer with PE-labeled anti-IFN-γ and PE-Cy7-labeled anti-TNF-α antibodies (eBioscience Inc).
To perform suitable data acquisition and analysis, autofluorescence, single-stained and FMO (fluorescence minus one) controls were included. Stained cells were passed through the BD FACSaria II flow cytometer.

Quaternized chitosan (Degree of quaternization DQ ≈ 40%) was synthesized according to our previous study [13]. α, β‐Glycerohosphate (GP) was bought from Kaiyuan pharmaceutical & Chemical (Shanxi, China). White oil was provided by Petro‐Canada Lubricants Inc. (Ontario, Canada). PO500 and Tween80 were from Sakamoto Yakuhin Kogyo (Osaka, Japan) and Merck & Co. Inc. (Darmstadt, Germany), respectively. Membrane emulsification equipment and porous membranes were obtained from Zhongke Senhui Microsphere Technology (Suzhou) (Jiangsu, China). Both Cy5 mono‐reactive NHS Esters and DiI were purchased from Fanbo Biochemicals (Beijing, China).
Inactivated PRRS antigens were kindly provided by Luoyang Huizhong Animal Medicine. FMD antigenic VLPs were from Lanzhou Veterinary Research Institute. APC‐labeled anti‐mouse CD4 antibody, PE‐labeled anti‐mouse CD69 antibody, FITC‐labeled anti‐mouse CD8 antibody, Percp‐Cyanine5.5‐labeled anti‐mouse CD44 antibody and eFluor450‐labeled anti‐mouse CD62L antibody, ELISA kits for IL‐4 and IFN‐γ were purchased from eBioscience (California, USA). ELISPOT kits were obtained from Abcam (England). CCK8 kits were from Dojindo Laboratories (Japan). Other materials were all of analytical grade.