Intracellular cytokine expression of PBMC by flow cytometry

PBMCs were stimulated for 4 h with phorbol 12-myristate 13-acetate (PMA; 100 ng/ml; Sigma-Aldrich) and ionomycin (1 μg/ml; Sigma-Aldrich) in the presence of 1× monensin (BioLegend, UK). After incubation, cells were stained with fluorochrome-labelled antibodies (anti-human CD3-FITC (BD Biosciences), CD3-PE-Cy7, IL-17A-PE, IL-4-APC, IL-6-APC (eBiosciences, San Diego, USA ), IFNγ-APC-Cy7, IL-8-PE, IL-10-Brilliant violet 421 and CD11b-APC-Cy7 (BioLegend, London, UK)).

Briefly, PBMC were washed twice with FACS buffer (300 g for 5 min at 4 °C) and resuspended at 10 × 10⁶ cells/ml; 20 μl (2 × 10⁵ cells) were dispensed per FACS tube and incubated with 5 μl Human TruStain FcX (Fc Receptor blocking solution; BioLegend) for 5 min at RT. The cells were then incubated with cell surface antibodies in a total volume of 100 μl FACS buffer for 30 min in the dark at 4 °C. After staining, cells were washed twice with FACS buffer and then fixed and permealized using the Foxp3 Transcription factor Staining Buffer Set (eBiosciences) according to the manufacturer’s instructions. Samples were then incubated with 5 μl Human TruStain FcX (Fc Receptor blocking solution; BioLegend) followed by incubation with intracellular cytokine antibodies in a total volume of 100 μl permealization buffer (eBiosciences) for 40 min in the dark at 4 °C. Cells were washed and acquired on the FACSCantoII flow cytometer (BD Biosciences). Data analysis was performed blindly using FlowJo software version 10.07 for Windows (Tree Star, Oregon, USA).

Gating was performed by first dividing PBMCs into CD11b⁺CD3⁻, CD11b⁻CD3⁺ and CD11b⁻CD3⁻ cells (Fig. 2). Gates for intracellular cytokines IL-4, IL-6, IL-8, IL-10, IL-17A and IFNγ were set on total live cells based on the unstained control, and the same gates were then applied to CD11b⁺CD3⁻, CD11b⁻CD3⁺ and CD11b⁻CD3⁻ cell subsets.

IL-8 producing PBMCs under non-stimulated conditions and after stimulation with PMA/ionomycin. Percentage of total IL-8 producing PBMCs from controls and nAMD patients (a) and composition of IL-8 producing cells from all samples (b). Percentage of CD11b⁺CD3^- cells that were positive for intracellular IL-8 from controls and nAMD patients (c). Controls n = 27, nAMD = 28; mean + SEM; #P < 0.05, ##P < 0.01 in univariate analysis suing independent samples t test; *P < 0.05 in multivariate analysis using multinomial logistic regression