Model 3550 uv elisa reader

2×10⁴ cells were seeded in 96-well plates, and, the day after, starved in KBM. Culture stimulation with IL-22 was conducted either in the presence or absence of 12.5 µM DCE, CS or HCS. After 2 d of treatment, cells were stained with 0.5% crystal violet, whose incorporation was measured at 540 nm in an ELISA reader (model 3550 UV ELISA reader; Bio-Rad, Hercules, CA).

8 × 10⁴ cells were seeded in 12-well plates and, the day after, starved in KBM. Culture stimulation with IFN-γ, IL-22, or TNF-α was conducted either in the presence or absence of tofacitinib. After 2 d of treatment, cells were evaluated by Trypan blue exclusion test. Crystal violet assays were also performed to evaluate proliferation. Thus, 2 × 10⁴ cells were grown for 48 h in 96-well plates and stained with 0.5% crystal violet, whose incorporation was measured at 540 nm in an ELISA reader (model 3550 UV ELISA reader; Bio-Rad, Hercules, CA).

SOCS1- and SOCS3 stably transfected keratinocytes or cultured keratinocytes previously transfected with SOCS3 or control siRNA were seeded in 96-well plates (1.5–2.0 × 10⁴ cells/well), and, the day after, starved in medium without serum. At 60% confluence, cells were treated with 75 ng/ml of IL-22 for 3 days, stained with 0.5% crystal violet (Bio-Rad, Hercules, CA, USA), and then analyzed in an ELISA reader (model 3550 UV ELISA reader; Bio-Rad). For proliferation analysis of healthy and transformed keratinocytes upon peptide or chemical inhibitor treatment, 1 × 10⁴ healthy keratinocytes, CAL27 and CAL33 cells were plated in 96-well plates in quadruplicate for each condition. After 1 day, medium was changed with fresh medium in the presence or not of Ctrl1 or KIR-ESS peptides (40 μM final concentration) or 10 μM STAT3 (S3I-201, Santa Cruz) or 20 μM Erk1/2 (PD98059, Calbiochem). After 1-h pre-incubation, cells were treated with IL-22 or left unstimulated, cultured for other 24, 48 and 72 h, and the number of viable cells determined by CyQUANT Cell Proliferation Assay, accordingly to manufacture's protocol. Fluorescence intensities were detected by using a VICTOR3 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA).