Fixation permeabilization solution kit

Freshly isolated splenocytes were resuspended at a density of 5 × 10⁶ cells/mL in complete RPMI and following an overnight rest at 4 °C, 1 × 10⁶ cells per well were distributed into v-bottom 96-well plates. Cells were pelleted and resuspended in 100 μL of complete RPMI containing an overlapping peptide pool containing 316 peptides (each 15 amino acids in length, 11 amino acid overlap) spanning the SARS-CoV-2 spike antigen, at a final concentration of 0.5 μg/mL per peptide (Genscript). A second well with DMSO only was used as a negative control for each sample. After 1 h of incubation at 37 °C, Brefeldin A (Biolegend) was added to a final concentration of 5 μg/mL and cells were incubated for an additional 4 h. Following stimulation, cells were washed with PBS and stained with fixable viability dye (eBioscience, e506). Extracellular staining was performed in FACS buffer (PBS + 2% FBS + 2 mM EDTA). Cells were then washed, fixed, and permeabilized with the eBiosciences Fixation/Permeabilization Solution Kit. Intracellular staining was then performed in permeabilization. Samples were collected on a Cytoflex LX (Beckman Coulter). Analysis of flow cytometry data was performed using FlowJo software (version 10.7.1).

Cells were incubated with surface markers against pre-conjugated antibodies CD45-APC (ebioscience, San Diego, CA, USA) and CD86-FITC (BD bioscience, San Jose, CA, USA) for 20 min. Cells were then permeabilized for intracellular staining with a fixation/permeabilization solution kit (ebioscience, San Diego, CA, USA). Cells were then separated into two groups and incubated with markers CD206 (Abcam, Cambridge, MA, USA) or TNFα (BD bioscience, San Jose, CA, USA) and TMEM119 (Novus, Centennial, CO, USA). Secondary antibodies PE (eBioscience, San Diego, CA, USA) and PerCP (BD bioscience, San Jose, CA, USA) were used in both groups. Cells were then washed and analyzed using the Cytoflex (Beckman Coulter, Indianapolis, IN, USA).

Cells were incubated with surface markers against pre-conjugated antibodies CD45-APC (eBioscience, San Diego, CA) and CD86-FITC (BD bioscience, San Jose, CA) for 20 min. Cells were then permeabilized for intracellular staining with a fixation/permeabilization solution kit (eBioscience, San Diego, CA). Cells were then separated into two groups and incubated with markers CD206 (Abcam, Cambridge, MA), TNFα (BD bioscience, San Jose, CA), and IL-10 (BD bioscience, San Jose, CA), or TNFα (BD bioscience, San Jose, CA) and TMEM119 (Novus, Centennial, CO). Secondary antibodies PE (eBioscience, San Diego, CA) and PerCP (BD Bioscience, San Jose, CA) were used in both groups. Cells were then washed and analyzed using the Cytoflex (Beckman Coulter, Indianapolis, Indiana).

Cells were stained with PE-Cy7 anti-F4/80, PE-Cy7 anti-CD11c, PerCP anti-CD3, PE anti-PD-L1, APC anti-CD86, FITC-CD206, PE anti-Foxp3, APC anti-CD25, and APC anti-IFNγ (BioLegend, San Diego, CA). Labile cell iron was measured using 10 μM calcein acetoxymethyl (calcein_AM, Thermo Fisher Scientific, MA). Lipid peroxidation was assessed by C11-BODIPY 581/591 (Thermo Fisher Scientific, MA), and the amount of ROS was measured by a DCFDA cellular ROS detection assay kit (Abcam, Cambridge, UK). CD4⁺IFNγ⁺ cells were detected by an intracellular staining kit (Fixation/Permeabilization Solution Kit, Franklin Lakes, NJ), and CD4⁺CD25⁺Foxp3⁺ cells were measured using a Foxp3/transcription factor staining buffer set (eBioscience). All data were analyzed using FACS LSR Fortessa cytometry with BD CELL Quest Pro software.