Fixable aqua dead cell kit

Splenocytes from vaccinated mice were cultured at 37°C with 5% CO₂ for 18 hrs in the presence of a megapool of BG505 SOSIP-specific peptides at a concentration of 2.5μg/mL per peptide (Cat: ARP-13123, NIH HIV reagent program) in 96-well U-bottom plates at 1 × 10⁶ splenocytes per well. A stimulation condition with an equal percentage amount of DMSO was included as a negative control, while PMA/Ionomycin (eBioscience, Cat: 00-4978-93) was used as a positive control. Following stimulation, cells were stained with live/dead Fixable Aqua Dead Cell Kit (Cat: L34957; Invitrogen) and anti-mouse CD16/32 (Clone: 93; Cat:101319; BioLegend) for 20 mins at 4°C, protected from light. Cells were then surface stained for 45 mins at 4°C protected from light with the following fluorochrome-conjugated anti-mouse Abs: CD3 (Clone: 17A2, Cat: 100216), CD4 (Clone: GK1.5; Cat:100414), CD8 (Biolegend, Clone: 53 – 6.7, Cat: 100748), CD25 (Clone: PC61; Cat: 102010), OX40 (Clone: OX-86; Cat:119411), and PD-L1 (Clone: 10F.9G2; Cat: 124321) from BioLegend. Samples were acquired on a BD LSR Fortessa and data were analyzed using FlowJo software v.10 (BD Life Sciences).

Single cell suspensions of splenocytes from vaccinated mice were cultured for 6 hours in the presence of five SARS-CoV-2 spike peptide pools [5 ug/mL final concentration] or in a 1x working dilution of Cell Activation Cocktail (BioLegend) (Cat: 423301) in 96-well U bottom plates at a concentration of 10⁶ cells per well. As a negative control, cells were treated with an equal percentage amount of DMSO. All samples were also treated with a protein transport inhibitor (eBioscience) (Cat: 00-4980-93) for the duration of the 6 hour stimulation. Following stimulation, cells were stained with the live/dead Fixable Aqua Dead Cell Kit (Invitrogen) (dilution 1/100; Cat: L34957) to gate on live cells and anti-mouse CD16/32 antibody (BioLegend) (Clone: 93; Cat: 101319). Cells were then stained at 4°C for 30 minutes with the following fluorochrome-conjugated anti-mouse antibodies: CD4 (Clone: GK1.5; Cat: 100438), CD8 (Clone: 53-6.7; Cat: 100714), IL-4 (Clone: 11B11; Cat: 504133), IFN-γ (Clone: XMG1.2; Cat: 505810), TNF-α (Clone: MP6-XT22, Cat: 506346), CD3 (Clone: 145.2C11; Cat: 100310), PD-1 (Clone: RMP1-30; Cat: 109110), CXCR5 (Clone: 2G8; Cat: 560615), CD154 (Clone: MR1 Cat: 106510) FoxP3 (Clone : MF-14 Cat: 126422). All cytometric analyses were performed using an LSR II flow cytometer (BD), and data were analyzed using FlowJo software (Treestar).

NK92 cells were first stained using the Fixable Aqua Dead Cell Kit (Thermofisher), followed by incubation with either with titrated amounts of Darzalex (Daratumumab) (Johnson & Johnson), or plasma from patients or healthy control and incubated for 10 mins at room temperature. The cells were then washed using MACS buffer (0.5% BSA + 2mM EDTA in PBS) followed by staining with different clones of anti-CD38: LS198-4-2 (Beckman Coulter), JK36 (Beckman Coulter) and HIT2 (BD Biosciences) for 10 mins in the dark at room temperature. The cells were then washed and analyzed using BD LSRFortessa™ cell analyzer. Data analyses were performed using Flowjo V10.5.3 (BD).