Alexa fluor 488 igg

The cell density was adjusted to 1 × 10⁶/100 μl/flow tubes, and 20 μl CD4 PE-Cy5/CD25 PE cocktail antibody (Thermo Fisher Scientific) was added to each tube. After thoroughly mixing, the cells were incubated at room temperature in dark for 20 min. After the cells were washed, the supernatant was discarded after centrifugation at 250 ×g for 5 min. Then, 1 ml of fixation/permeabilization working solution (BD Pharmingen, NJ, United States) was added and incubated in dark at room temperature for 20 min, followed by centrifugation at 250 ×g for 5 min. Then the supernatant was discarded. The cells were washed with 1 ml permeabilization buffer (Thermo Fisher Scientific), centrifuge at 250 ×g for 5 min and further discard the supernatant. Subsequently, the cells were resuspended with 100 μl permeabilization buffer (Thermo Fisher Scientific) and 5 μl Alexa Fluor® 488 Foxp3 antibody (Thermo Fisher Scientific) or 5 μl Alexa Fluor® 488 IgG (Abcam) was added to each tube and incubated at room temperature in dark for 30 min. Ultimately, the cells were washed twice with cell staining buffer and were resuspended with 0.5 ml of cell staining buffer for detection on flow cytometry BD FACSCanto II (BD, Franklin Lakes, NJ, United States).

Cells were fixed with 4% paraformaldehyde in PBS for 20 minutes and were then permeabilized and blocked for 1 hour with 0.3% Triton X-100, 2% bovine serum albumin, 5% donkey serum and 5% goat serum. The Spike S primary antibody (1:100, Sino Biologicals Cat# 40,150-R007) was added to samples overnight at 4°C. Samples were rinsed 5 times for 2 minutes each with PBS containing 0.3% Triton X-100. The goat anti-rabbit Alexa Fluor® 488 IgG (Abcam, Cat# ab150077) secondary antibody was diluted 1:1000 in blocking buffer and was then added for 2 hours at room temperature. Samples were then rinsed 5 times for 2 minutes each with PBS containing 0.3% Triton X-100, followed by DAPI diluted in PBS at 1:5000 for 10 minutes. Images were obtained using Zeiss LSM880 confocal microscope using the Airyscan detector and a 20X air objective (Carl Zeiss GmbH, Jena, Germany) and were quantified using CellProfiler 2.0. Alternatively, plates were imaged using the Operetta (PerkinElmer, Waltham, MA) system.