Cytoflex lx platform

Measurements were performed on the CYTOFLEX LX platform of Beckman Coulter. The generated data were analyzed using the FlowJo Software Version 10.6.1 which also was used to generate the cytometry graphics.
Single-cell solutions containing 5 × 10⁵ cells were used for each flow cytometric staining. Staining was performed in a 96-well plate (TC Plate 96 Well Suspension, R [Sarsted]). Live- dead staining was performed using Zombie UV in a dilution of 1:1000 (Zombie UV™ Fixable Viability Kit [BioLegend]) to exclude dead cells. Subsequently, binding cells were blocked with 0.5 µL of Fc receptor-blocking antibodies (TruStain FcX™ [anti mouse CD16/32 Isotype Rat igG2a, λ clone: 93 Biolegend]) to reduce non-specific antibody binding.
To identify DC and T cells, samples were incubated for at least 15 min with the appropriate antibody mixtures. After surface staining, a FoxP3 intracellular staining was performed to mark Tregs, as recommended by the manufacturer.

For cytofluorimetric analyses, 1–2 × 10⁵ cells/treatment groups were treated for 24 h and were fixed in 4% PFA for 10 min at RT. The cells were then washed twice with 1X TBS and permeabilized in absolute methanol for 10 min, and then washed once in 1X TBS buffer containing 1% FBS, 2 mM EDTA, 0.1% NaN₃. For intercellular total JNK and phospho-JNK expression, cells were respectively incubated with Anti-JNK1 + JNK2 + JNK3 (Abcam, #ab179461, Toronto, ON, Canada) or Anti-JNK1 + JNK2 + JNK3 phospho (T183 + T183 + T221) (Abcam, #ab124956, Toronto, ON, Canada) antibodies (1:100) in TBS buffer for an hour at RT. FITC-conjugated normal rabbit IgG1 (1:250) (Invitrogen, #A21246, Life Technologies, Eugene, OR, USA) secondary antibody was used for 30 min incubation at RT in the dark and phospho and total JNK were detected independently in all treatment groups, respectively. The CytoFLEX LX Platform (Beckman Coulter Life Sciences, Mississauga, ON, Canada) was used for sample acquisition (10,000 events/sample). Data were collected and then analyzed on CytExpert software (Beckman Coulter, Brea, CA, USA).