Cytofix cytoperm buffer set

Thawed PBMC were washed twice with complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l‐glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, 55 μM β‐mercaptoethanol) plus 0.02 mg/ml DNAse. PBMCs were stimulated for 4 h at 37°C in a 5% CO2 atmosphere with PMA (100 ng/ml) and Ionomycin (1 μg/ml) in complete culture medium. For each sample, at least 2 million cells were left unstimulated as negative control, and 2 million cells were stimulated. All samples were incubated with a protein transport inhibitor containing brefeldin A (Golgi Plug, Becton Dickinson). After stimulation, cells were stained with LIVE‐DEAD Aqua (Thermo Fisher Scientific) and surface mAbs recognizing HLA‐DR‐PE‐Cy7, CD14‐APC, and CD16‐BV421 (BioLegend, San Diego, CA, USA). Cells were washed with stain buffer, fixed, and permeabilized with the cytofix/cytoperm buffer set (Becton Dickinson) for cytokine detection. Then, cells were stained with previously titrated mAbs recognizing IFN‐γ‐FITC and TNF‐BV605 (all mAbs from BioLegend). Samples were acquired on Attune NxT acoustic cytometer (Thermo Fisher).

For functional assays on cytokine production by T cells, thawed isolated PBMCs were stimulated for 16 h at 37 °C in a 5% CO₂ atmosphere with anti-CD3/CD28 (1 μg/mL) in complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, 55 μM β-mercaptoethanol). For each sample, at least 2 million cells were left unstimulated as negative control, and 2 million cells were stimulated. All samples were incubated with a protein transport inhibitor containing brefeldin A (Golgi Plug, Becton Dickinson) and previously titrated concentration of CD107a-PE. After stimulation, cells were stained with LIVE-DEAD Aqua (ThermoFisher Scientific) and surface mAbs recognizing CD3 PE- Cy5, CD4 AF700, and CD8 APC-Cy7 (Biolegend, San Diego, CA, USA). Cells were washed with stain buffer, and fixed and permeabilized with the cytofix/cytoperm buffer set (Becton Dickinson) for cytokine detection. Cells were next stained with previously titrated mAbs recognizing IL-17 BV421, TNF BV605, IFN-γ FITC, IL-2 APC, or granzyme-B BV421 (all mAbs from Biolegend). Then, a minimum of 100,000 cells per sample were acquired on a Attune NxT acoustic cytometer (ThermoFisher).

For functional assays on cytokine production by T cells, thawed isolated PBMCs were stimulated for 16 h at 37 °C in a 5% CO2 atmosphere with anti-CD3/CD28 (1 μg/mL) in complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, and 55 μM β-mercaptoethanol). For each sample, at least 2 million cells were left unstimulated as negative control, and 2 million cells were stimulated. All samples were incubated with a protein-transport inhibitor containing brefeldin A (Golgi Plug, Becton Dickinson) and previously titrated concentration of CD107a-PE. After stimulation, cells were stained with LIVE-DEAD Aqua (ThermoFisher Scientific) and surface mAbs recognizing CD4 AF700, and CD8 APC-Cy7 (Biolegend, San Diego, CA, USA). Cells were washed with stain buffer and fixed and permeabilized with the cytofix/cytoperm buffer set (Becton Dickinson) for cytokine detection. Cells were next stained with previously titrated mAbs recognizing CD3 PE-Cy5, IL-17 BV421, TNF BV605, IFN-γ FITC, IL-4 APC, or granzyme-B BV421 (all mAbs from Biolegend). Then, a minimum of 100,000 cells per sample were acquired on a Attune NxT acoustic cytometer (ThermoFisher)^{53 (link)}. FCS data were acquired in list mode by Attune nxT Software v4.2. mAbs used are listed in Sup Data 6.