Cyto x cytoperm solution

1. CD45BV421/CD34PE-Cy7 2.
CD45BV421/CD56+CD16PE/CD14PerCP/CD19APC-H7/CD3V500/CD34PE-Cy7 3.
CD45BV421/CD56+CD16PE/CD14PerCP/CD19APC-H7/CD3V500/CD34PE-Cy7 4.
CD45BV421/CD56+CD16PE/CD14PerCP/CD19APC-H7/CD3V500/CD34PE-Cy7
Table 7. Panel of surface labelling markers.
The samples were incubated at room temperature in the dark for 30 minutes. Then, the erythrocytes were lysed with 2 ml of FACS lysing solution (BD Biosciences) and incubated for 15 minutes under the same conditions as the previous incubation. After two washes with PBS and centrifugation at 500 × g for 5 minutes, the cell pellets were resuspended in 500 µl of BD Cyto x/Cytoperm™ solution (BD Biosciences) to x and permeabilize the cells. The cells were incubated in the dark for 20 minutes at room temperature.
Then, the cells were centrifuged at 500 x g for 5 minutes again. Cell pellets were resuspended in freshly prepared Perm/Wash Buffer and incubated for 10 minutes in the dark. Then, the cells were centrifuged, the supernatant was removed, and the pellets were treated by primary antibodies to the scheme shown in Table 8.

Cryopreserved PBMCs were rapidly thawed, washed in R10 media (RMPI-1640 + 10% FBS + 1% Pen/Strep), and 10 6 cells added to wells of a 96-well U-bottom plate. Cells were stimulated with 2ug/ml overlapping S1 or M peptide pools, or left unstimulated, for 2 hr at 37°C, 5% CO2. Anti-CD107a-BV785 (1:100 dilution, clone H4A3), anti-CD28 (clone CD28.2) and anti-CD49d (clone R1-2) were added at 1ug/ml to all wells at the time of peptide addition. After 2 hr, brefeldin A (5ug/ml) and monensin (2uM) were added, and cells were incubated at 37°C, 5% CO2 for an additional 16 hr. After stimulation, cells were washed two times with FACS buffer (PBS + 1mM EDTA + 0.05% BSA). Surface staining was performed at 4 °C for 30 mins. Cells were then washed two times in FACS buffer, and xed and permeabilized at 4 °C for 30 mins using BD Cyto x/Cytoperm solution. Cells were then washed twice with 1x BD Perm/Wash buffer, and stained for intracellular markers at 4°C for 30 mins. Two further washes with 1x BD Perm/Wash buffer were performed and cells were stored in FACS buffer at 4°C. Samples were acquired on a custom Cytek Aurora spectral analyzer (4 laser; UV, violet, blue, and red) using SpectroFlo v2.2. Data were analyzed using FlowJo v. 10.6.2 and Prism v. 8.3.0.

Cells were xed in BDCyto x/Cytoperm solution (BD Biosciences, NJ, USA) and permeabilised with 0.1% Triton-X. The cells were incubated overnight with primary antibody for nuclear factor kappa B (NF-κB). Cell Signaling Technology Inc., Danvers, USA. cst 8242s, 1:500) followed by labelling with FITC goat antirabbit IgG (Wuhan Servicebio Technology Co., Ltd, Wuhan, China. 1:400). Later the cells were incubated with 0.5 mg/mL DAPI for staining the nuclei. Images were obtained using a uorescence microscope. To analyse the ratio of nuclear and cytoplasmic immune uorescence of NF-κB, the percentage of cells showing high uorescence in different areas of a cell (either nucleus or cytoplasm) was counted. A total of 200 cells were counted per group. Densitometry analysis of the nuclear translocation of NF-κB was expressed as a percentage of total cells.