Expo32

Leukocytes were prepared from lymph nodes draining the inflamed joint, spleen, and thymus of NI and CIA mice. Synovial cells were harvested by digesting synovial tissues with collagenase. The cells were incubated with primary antibodies for 15 min at 4°C. Antibodies against CD4, CD8, CD44, Foxp3, B220, and CD11c were purchased from eBioscience; the anti-CD62L antibody was from BD Pharmingen. Nonspecific staining was ascertained using isotype-matched control antibodies. T_H cells were fixed in fixation/permeabilization buffer for 30 min; resuspended in 100 µl of permeabilization buffer; incubated for 30 min at 4°C with Alexa 488- or phycoerythrin (PE)-conjugated anti-IFNγ (eBioscience), fluorescein isothiocyanate (FITC)-conjugated anti-IL4, PE-conjugated anti-IL17A or isotype control antibodies (eBioscience); and analyzed by flow cytometry using EPICS XL and EXPO32 software (Beckman Coulter).

U937 cells were seeded in 6-well plates in a final concentration of 7 × 10⁵ cells per well in a final volume of 3 mL/well of RPMI with 10% FBS, 100 U·mL⁻¹ Penicillin, 100 μg·mL⁻¹ Streptomycin and 1 mM Na Pyruvate, and incubated overnight at 37 °C. The following day, cells were incubated with extracts from raw and 3-day germinated mung bean (both 10× diluted) and ethanol control (1%) for 24 h, followed by a 16 h-treatment with 1 mmol·L⁻¹ PQ/0.7 mmol·L⁻¹ SNAP as final concentration as previously described for the HT-29 cell line. After treatment, cells were divided into two aliquots, one for ROS determination and the other for RNA extraction and gene expression studies. The cells were washed twice with PBS by centrifugation and discarding the supernatant, prior to their incubation with 1 µM acetic 5-(chloromethyl)-2-(3,6-diacetoxy-2,7-dichloro-9H-xanthen-9-yl) benzoic anhydride (CM-H2DC-FDA) dye for 45 min at 37 °C. ROS determination was performed using a Beckman Coulter Epics XL-MCL flow cytometer (Beckman Coulter, Fullerton, CA, USA) and analysis software Expo 32 (Beckman Coulter, Fulleton, CA, USA). Experiments were carried out 3 times independently with duplicates for each treatment in each experiment.