Live dead aqua fixable cell stain

Trogocytosis and ADCC were measured using our previously published assay [43 (link)]). Briefly, CEM.NKR.CCR5 cells (NIH AIDS Reagent Program, Division of AIDS, NIAD, NIH courtesy of Dr. Alexandra Trkola [44 (link)]) were washed with PBS and stained with PKH26 (Sigma-Aldrich, St-Louis, MO, USA) at 2 μM in Diluant C at room temperature for 5 minutes. Cells were then washed with RPMI 10% FBS (R10), resuspended in R10, and incubated with gp120 (Case A2) for 1 hour at room temperature. Cells were then washed twice with R10 and incubated with diluted plasma samples for 1 hour at room temperature. Effector cells (PBMC form one healthy subject) were then added in R10 at an effector to target cell ratio of 50:1 and incubated for 5 hours at 37°C. After the incubation, cells were washed, stained with Live/dead aqua fixable cell stain (Life Technologies, Eugene, OR, USA) for 15 minutes at room temperature, washed, and fixed with 2% Formaldehyde (Tousimis, Rockville, MD) for 15 minutes at room temperature. Data were acquired on a BD LSRII instrument (BD Biosciences) and analyzed using FlowJo Version 9.9.6 software (TreeStar, Ashland, OR, USA).

Cryopreserved PBMC specimens were thawed and washed, and counts and viability were assessed using the Countless Automated Cell Counter system (Invitrogen, Carlsbad, CA). Cells were washed and stained in Brilliant Violet Stain Buffer (BD Biosciences, San Jose, CA) at room temperature for 15 min in 96-well V-bottom plates in the dark. Samples were then washed and fixed using Cytofix/Cytoperm (BD Biosciences) before flow cytometry data acquisition. Intracellular staining for IFNγ and promyelocytic leukaemia zinc finger (PLZF) were performed in Perm/Wash (BD Biosciences) at room temperature for 15 min in the dark. mAbs used in flow cytometry: CD3 AF700, CD3 PerCP-Cy5.5 (both clone UCHT1), CD4 BV605 (clone RPA-T4), CD8 BV711 (clone RPA-T8), CD38 APC-H7 (clone HB7), CD69 AF00 (clone FN50de), CD161 BV421 (clone DX12), CCR6 BV786 (clone 11A9), HLA-DR APC (clone L243), IFNγ APC (clone B27), and PD-1 PE-Cy7 (clone EH12.1) were all from BD Biosciences, TCR Vα7.2 PE, TCR Vα7.2 PerCP-Cy5.5 (clone 3C10) were from Biolegend (San Diego, CA, USA), and PLZF APC was from R&D Systems (Minneapolis, MN). Live/dead aqua fixable cell stain was from Life Technologies (Eugene, OR, USA) and added to the cells together with the cell surface antibodies. Data were acquired on a BD LSRFortessa instrument (BD Biosciences) and analyzed using FlowJo Version 9.8.5 software (TreeStar, Ashland, OR, USA).

Cryopreserved specimens were thawed and washed, and counts and viability were assessed using the Countess Automated Cell Counter system (Invitrogen, Carlsbad, CA, USA). Cells were washed and stained in Brilliant Violet Stain Buffer (BD Biosciences, San Jose, CA, USA) at room temperature for 15 min in 96-well V-bottom plates in the dark. Samples were then washed and fixed using Cytofix/Cytoperm (BD Biosciences) before flow cytometry data acquisition. Intracellular staining was performed in Perm/Wash Buffer (BD Biosciences). Monoclonal antibodies used in flow cytometry: CD3 AF700, CD3 PerCP-Cy5.5 (both clone UCHT1), CD4 BV605 (clone RPA-T4), CD8 BV711 (clone RPA-T8), CD161 BV421 (clone DX12), CCR6 BV786 (clone 11A9), HLA-DR APC (clone L243), IFNγ APC (clone B27), and PD-1 PE-Cy7 (clone EH12.1) were all from BD Biosciences, TCRα24 FITC (clone C15) and TCR Vβ11 PE (clone C21) were from Beckman Coulter (Indianapolis, IN, USA), and TCR Vα7.2 PercP-Cy5.5 (clone 3C10) was from BioLegend (San Diego, CA, USA). Live/dead aqua fixable cell stain was from Life Technologies (Eugene, OR, USA). Data were acquired on a BD LSRFortessa instrument (BD Biosciences) and analyzed using FlowJo Version 9.8.5 software (TreeStar, Ashland, OR, USA).

Trogocytosis was measured using a previously described assay (40 (link)). Briefly, SARS-CoV-2 S-expressing expi293F cells were stained with PKH26 (Sigma-Aldrich). Cells were then washed with and resuspended in R10 media. Cells were then incubated with 200-fold diluted plasma samples for 30 min at 37 °C. Effector PBMCs were next added to the R10 media at an effector to target cell ratio of 50:1 and then incubated for 5 h at 37 °C. After the incubation, cells were washed, stained with live/dead aqua fixable cell stain (Life Technologies), and CD14 APC-Cy7 (clone MϕP9) for 15 min at RT, washed again, and fixed with 4% formaldehyde (Tousimis) for 15 min at RT. Fluorescence was evaluated on an LSRII flow cytometer (BD Bioscience). Trogocytosis was evaluated by measuring the PKH26 mean fluorescence intensity of the live CD14+ cells.

Cryopreserved specimens were thawed and washed, and counts and viability were assessed using the Countess Automated Cell Counter system (Invitrogen, Carlsbad, CA). Cells were washed and stained in Brilliant Violet Stain Buffer (BD Biosciences, San Jose, CA) at room temperature for 15 min in 96-well V-bottom plates in the dark. Samples were then washed and fixed using Cytofix/Cytoperm (BD Biosciences) before flow cytometry data acquisition. Intracellular staining was performed in Perm/Wash (BD Biosciences). mAbs used in flow cytometry: CD3 AF700, CD3 PerCP-Cy5.5 (both clone UCHT1), CD8 BV711 (clone RPA-T8), CD38 APC-H7 (clone HB7), CD127 FITC (clone HIL-7R-M2), CD161 BV421 (clone DX12), CCR6 BV786 (clone 11A9), HLA-DR APC (clone L243), IFNγ APC (clone B27), and PD-1 PE-Cy7 (clone EH12.1) were all from BD Biosciences, PLZF APC was from R&D Systems (Minneapolis, MN), EOMES FITC (clone WD1928) was from eBioscience and TCR Vα7.2 PE (clone 3C10) was from Biolegend (San Diego, CA, USA). Live/dead aqua fixable cell stain was from Life Technologies (Eugene, OR, USA). Data were acquired on a BD LSRFortessa instrument (BD Biosciences) and analyzed using FlowJo Version 9.8.5 software (TreeStar, Ashland, OR, USA).