Zombie red viability marker

Two panels of fluorochrome-conjugated antibodies (BioLegend, San Diego, CA, USA) were used to identify the innate immunity cell populations and to analyze the activation marker expression: (1) CD11b-PE/Cy7, CD11c-PE, MHCII-Alexa488, CD103-PerCP-Cy5.5, CD45-APC/Cy7, CD64-BV421, CD24-BV510; (2) CD45-APC/Cy7, MHCII-Alexa488, Ly6G-PerCP-Cy5.5, CD86-BV421, CD83-BV510. T-cells staining was performed using the fluorochrome-conjugated antibody set containing CD4-PerCP-Cy5.5, CD8-PE/Cy7, CD62L-APC/Cy7, and CD44-BV421 (BioLegend, San Diego, CA, USA). Intracellular production of cytokines was assessed using antibodies against IFNγ-FITC, IL2-PE, and TNFα-BV510 (BioLegend, San Diego, CA, USA). Staining for the detection of intracellular markers was performed using the Fixation and Permeabilization Solution reagent kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Zombie Red viability marker (BioLegend, San Diego, CA, USA) was used to identify the dead cells. True Stain reagent (BioLegend, San Diego, CA, USA), containing antibodies to CD16/CD32, was added during the surface markers staining to block non-specific antibody binding. Data were collected on a Cytoflex flow cytometer (Beckman Coulter, Bray, CA, USA). The results were analyzed using the Kaluza Analysis 2.2 program (Beckman Coulter, Bray, CA, USA).

To estimate the percentage of CD4+ and CD8+ T lymphocytes producing cytokines after peptide stimulation, cells were stained with CD8-PE/Cy7, CD4-PerCP-Cy5.5, CD44-BV510, CD62L-APC/Cy7, IFNγ-FITC, TNFα-BV421, IL10-PE/Dazzle594, and IL2-PE antibodies (BioLegend, USA) using the BD Cytofix/Cytoperm™ Kit (BD Biosciences, USA) according to the manufacturer’s instructions. Zombie Red viability marker (BioLegend, USA) was used to identify dead cells. True Stain reagent, containing antibodies to CD16/CD32, was used to block non-specific antibody binding (BioLegend, USA). Data were collected on a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA). The results were analyzed using the Kaluza Analysis v2 software (Beckman Coulter, USA). Background values obtained from the non-stimulated cells were subtracted from the corresponding values of the stimulated samples before the statistical analysis to estimate the increase in the cytokine production levels upon peptide stimulation.

To analyze the innate immune cell populations, the panel of fluorochrome-conjugated antibodies was used, including CD11b-PE/Cy7, CD11c-PE, MHCII-Alexa488, CD103-PerCP-Cy5.5, CD45-APC/Cy7, CD64-BV421, and CD24-BV510. To estimate the percentage of CD8⁺ T-lymphocyte-producing cytokines after the peptide stimulation, cells were stained with CD8-PE/Cy7, CD4-PerCP-Cy5.5, CD44-BV510, CD62L-APC/Cy7, IFNγ-FITC, TNFα-BV421, and IL2-PE antibodies using the Fixation and Permeabilization Solution reagent kit (BD Biosciences) according to the manufacturer’s instructions. Zombie Red viability marker (BioLegend) was used to identify the dead cells. True Stain reagent, containing antibodies to CD16/CD32, was used to block nonspecific antibody binding (BioLegend). Data were collected on a CytoFlex flow cytometer (Beckman Coulter). The results were analyzed using the Kaluza Analysis v2 program (Beckman Coulter). To estimate the increase in the cytokine production levels upon the peptide stimulation, the background values obtained from the nonstimulated cells were subtracted from the corresponding values of stimulated samples before the statistical analysis.