Permeabilization buffer

Following stimulation, cells were stained for the desired surface markers as described above. The cells were then washed with cold PBS, and 500 μl of fixation buffer (Biolegend) was added to the tubes, briefly vortexed, and incubated in the dark at room temperature for 20 min. About 1 ml of Permeabilization buffer (Biolegend) was then added and samples were centrifuged at 458 × g for 5 min at 4 °C followed by one more wash using 1 ml of Permeabilization buffer. Cells were resuspended in 200 μl of Permeabilization buffer and cells were stained with antibodies against TNF-α, Ki67, Granzyme-B, IL-12, IL-6, and IFNγ or the respective isotype control overnight at 4 °C. Cells were then washed, filtered, and data were acquired using a flow cytometer.

Frozen PBMCs were thawed using complete RPMI (RPMI+10%FBS) and washed with PBS. The cells were then treated with cisplatin for 5 mins, followed by incubation with metal-conjugated surface antibodies cocktail (Table 1) for 30 mins at 37°C. Cells were washed twice with CyFACS buffer (PBS with 4% FBS, 0.05% sodium azide), followed by primary antibody staining for 30 mins on ice. Subsequently, cells were washed twice with CyFACS buffer, followed by permeabilization and fixation with Foxp3 fix/perm solution (eBioscience) for 30 mins on ice. Following this, cells were washed with permeabilization buffer (Biolegend) and then stained with intracellular antibody cocktail (Table 1) for 30 mins on ice, wash with Biolegend permeabilization buffer then stained with metal-conjugated streptavidin for 10 mins on ice. Finally, cells were washed with PBS and fixed overnight using 2% PFA made in PBS. The next day, cells were barcoded and stained with Cell-ID Intercalator-Ir (Fluidigm) in PBS for 20 mins at room temperature. Cells were washed twice with CyFACS buffer followed by a final wash using MiliQ water and passed through size filter. Filtered cells were analyzed using Helios mass cytometer (Fluidigm) with CyTOF software version 7.0.8493. Data analyses were performed using Flowjo V10.5.3 (BD) and Cytofkit2 (33 (link)).

Sera were added to enzyme-linked immunosorbent assay (ELISA) plates coated with RBD (300 ng/well; BEI NR-52309) (37°C 2 hours). CD16a.NK-92 cells (PTA-6967, American Type Culture Collection) with brefeldin A (Biolegend), Golgi Stop (BD Biosciences), and anti-CD107a (clone H4A3, BD Biosciences) were then added (37°C, 5 hours). Cells were stained with anti-CD56 (clone 5.1H11, BD Biosciences) and anti-CD16 (clone 3G8, BD Biosciences) and fixed (4% paraformaldehyde [PFA]). Intracellular cytokine staining to detect interferon-γ (IFN-γ; clone B27, BD Biosciences) and tumor necrosis factor-α (TNF-α; clone Mab11, BD Biosciences) was performed in permeabilization buffer (Biolegend). Markers were measured by flow cytometry as detailed above [8 (link)].

293 cells (Invivogen) were cultured in Eagle’s Minimum Essential Medium, (ATCC) with 10% heat inactivated FBS (Gibco) and 1% Penicillin/streptomycin (Gibco). Cells were transfected with NFAM1 cDNA bearing either an N-terminal or C-terminal Flag tag via the use of TransIT-293 transfection reagent (Mirus) diluted in Opti-MEM media (Gibco). 48 hours post transfection, cells were harvested using 0.05% Trysin-EDTA (Invitrogen). Cells were stained for 15 minutes with 0.1% Zombie NIR dye (BioLegend) diluted in PBS and washed twice. For detection of cell surface expression, cells were immediately stained with anti-FLAG clone L5 in PE (BioLegend), washed twice, and resuspended in fixation buffer (BioLegend). For detection of intracellular expression, cells were incubated in stabilizing fixative (BD) for 20 min at RT, washed twice and resuspended in permeabilization buffer (BioLegend) before being stained with anti-FLAG clone L5 in PE (BioLegend). Flow cytometry and analysis was performed as described above.

The cells were fixed and incubated at 4 °C for 20 min. After fixation, they were washed twice with a permeabilization buffer (BioLegend Cat#421002, San Diego, CA, USA). Next, the cells were resuspended in a master mix of intracellular-staining antibodies (anti-IL-13, eBioscience Cat#12-7133-82 PE, San Diego, CA, USA; anti-IL-5, BioLegend Cat#504305 APC) and incubated at 4 °C for 20 min. The cells were then washed twice with the permeabilization buffer, resuspended in a FACs buffer, and analyzed using a BD FACS Canto II flow cytometer and the FlowJo^TM v9 software.

NK cells were fixed and permeabilized with Fixation Buffer and Permeabilization Buffer purchased from Biolegend (San Diego, CA) and the procedures were conducted as instructed by the manufacturer.