Percp vio700

Stimulated cells were transferred to FACS tubes and washed. The cells were stained extracellularly for 10 min at room temperature with Viobility 405/452 Fixable Dye, antibodies against CD14, CD20, both conjugated to VioBlue (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD4 conjugated to PerCP (BD Biosciences). Subsequently, the stained cells were permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer’s instructions. Permeabilized cells were then stained intracellularly for 10 min at room temperature with antibodies against IL-2 conjugated to BV605 (BD Biosciences), TNF- α conjugated to PE-Vio 770, CD154 conjugated to PerCP-Vio 700, and IFN-γ conjugated to APC-Vio770 (Miltenyi Biotec) according to the methods of the supplier. Stained cells were quantified with a MACSQuant16^® analyzer flow cytometer (Miltenyi Biotec). FACS data were evaluated using FlowLogic software version 8.4 (Inivai Technologies, Victoria 3194 Australia). Reactive T cells were defined as CD4⁺ T cells expressing ≥2 of the following T_H1 activation markers: CD154, IFN-γ, IL-2 and TNF-α. DMSO background controls were subtracted from the data shown. The gating strategy used for all analyses can be found in the supplementary material (Figures S1 and S2).

Neutrophil isolation from whole human blood was done using MACSxpress Neutrophil Isolation Cocktail Kit (Miltenyi Biotec B.V. & Co. KG, Germany) according to the manufacturer’s instructions. Isolated cells were counted using Auto Hematology Analyzer BC-5300 (Mindray Medical Germany GmbH, Germany).
100 µl of isolated cells were mixed with antibodies against CD66b (neutrophils; 2 µl 1:4 diluted, Pacific blue-labelled; BioLegend, CA, USA), CD14 (monocytes; 1 µl undiluted, PerCP-Vio700-labelled; Miltenyi Biotec B.V. & Co. KG, Germany), CD3 (lymphocytes; 1 µl undiluted, VioGreen-labelled; Miltenyi Biotec B.V. & Co. KG, Germany) and CD45 (immune cells; 1 µl undiluted, PE-labelled; Miltenyi Biotec B.V. & Co. KG, Germany). After 10 min incubation at 4 °C, stained cells were washed with 1 ml cell wash by centrifugation for 5 min at room temperature (RT) and 300×g. The cell pellet was resuspended in 100 µl cell wash to analyze purity of the isolated cell fraction using FACS (BD FACSCanto II; BD Biosciences, San Jose, CA, USA). Using FACS analysis, we found that about 96–98% of cells were positive for CD66b indicating a very high amount of neutrophils in the obtained cells (data not shown). 2 × 10⁶ cells in RPMI with 5% heat-inactivated human serum (50 µl) were added to the surrounding medium of the skin samples.