Monoclonal antibodies (mAbs) to the following human proteins were used
for staining: CD3 (UCHT1), CD4 (RPA-T4), IFN-γ (4S.B3), IL-17 (BL168)
(Biolegend), phospho (p)-STAT1 (KIKSI0803), (all from eBioscience), STAT1
(246523; R&D Systems). Appropriate isotype controls were used in
parallel. PBMCs were incubated with mAbs against surface markers for 30 min on
ice. Intracellular staining with STAT1 mAb was performed using an eBioscience
Fixation/Permeabilization kit according to the manufacturer's
instructions. For p-STAT1 staining, PBMCs were stimulated for 20 min with
appropriate cytokines in complete medium, fixed with 2% paraformaldehyde
for 20 min on ice, permeabilized with 90% methanol for 30 min on ice and
stained using CD3, CD4 and p-STAT1 mAbs in PBS for 30 min. For cytokine
detection, cell suspensions were incubated with Phorbol myristate acetate (PMA)
(Sigma-Aldrich; 50 ng/mL), Ionomycin (Sigma-Aldrich; 500 ng/mL) and
GolgiPlug™ (BD Biosciences; according to manufacturer's
instructions) for 4 h in complete medium before surface staining.
Permeabilization and intracellular IFN-γ and IL-17 staining was carried
out using an eBioscience Fixation/Permeabilization kit as described above. Data
were collected with an LSRFortessa™ cytometer (BD Biosciences) and
analyzed with FlowJo software (Tree Star, Inc.).
for staining: CD3 (UCHT1), CD4 (RPA-T4), IFN-γ (4S.B3), IL-17 (BL168)
(Biolegend), phospho (p)-STAT1 (KIKSI0803), (all from eBioscience), STAT1
(246523; R&D Systems). Appropriate isotype controls were used in
parallel. PBMCs were incubated with mAbs against surface markers for 30 min on
ice. Intracellular staining with STAT1 mAb was performed using an eBioscience
Fixation/Permeabilization kit according to the manufacturer's
instructions. For p-STAT1 staining, PBMCs were stimulated for 20 min with
appropriate cytokines in complete medium, fixed with 2% paraformaldehyde
for 20 min on ice, permeabilized with 90% methanol for 30 min on ice and
stained using CD3, CD4 and p-STAT1 mAbs in PBS for 30 min. For cytokine
detection, cell suspensions were incubated with Phorbol myristate acetate (PMA)
(Sigma-Aldrich; 50 ng/mL), Ionomycin (Sigma-Aldrich; 500 ng/mL) and
GolgiPlug™ (BD Biosciences; according to manufacturer's
instructions) for 4 h in complete medium before surface staining.
Permeabilization and intracellular IFN-γ and IL-17 staining was carried
out using an eBioscience Fixation/Permeabilization kit as described above. Data
were collected with an LSRFortessa™ cytometer (BD Biosciences) and
analyzed with FlowJo software (Tree Star, Inc.).