Cytation 3 imaging reader

Cells were seeded at 200,000 cells per well of a six-well plate and transiently transfected with 1 μg of pPKA-SPARK using the PEI MAX 40K transfection reagent. Twenty-four hours after transfection, cells were treated with FI for specified time points. Fluorescence imaging and image analysis were done on live cells using the Cytation 3 Imaging Reader (Agilent) the software Gen5.

ATP levels were measured with the CellTiter-Glo (CTG) luminescent assay (Promega, no. G7572) according to the manufacturer’s instructions. Briefly, cells were seeded at a density of 70,000 cells per well of 96-well plates. After 24 hours, CTG reagent was added, and the luminescence was measured with the Cytation 3 Imaging Reader from Agilent. The luminescence of control cells was set to 1.

cAMP was measured using a cAMP-Glo assay kit (Promega, no. V1501) according to the manufacturer’s protocol. Briefly, cells were seeded at a density of 3000 cells per well of a white opaque 384-well plate with a clear bottom (Corning, no. 3765). After 24 hours, medium was replaced by serum-free medium containing FI and incubated for 30 min, 1 hour, 3 hours, and 6 hours. Cells were lysed using the cAMP-Glo lysis buffer by shaking at room temperature for 15 to 30 min. cAMP detection solution containing the enzyme PKA was added, mixed, and incubated for 20 min at room temperature. Then, the Kinase-Glo reagent was added, mixed, and incubated for 10 min at room temperature. Luminescence was measured using the Cytation 3 Imaging Reader from Agilent.

Cells were seeded into a 96-well plate 24 hours before drug treatments. A serial dilution ranging from 10⁻⁹ to 10⁻⁴ M for 4-OHT or MRT in DMEM medium without phenol red, containing 5% CHFBS, penicillin/streptomycin, l-glutamine, and E2 (5 pM), was prepared and added to the cells. For the combination of 4-OHT + MRT, 50 nM or 120 nM MRT was added to the serial dilutions of 4-OHT. For the combination of 4-OHT and purine nucleosides, purine-depleted serum was charcoal-treated for the additional depletion of hormones. After 7 days of 4-OHT, 4-OHT + MRT, or 4-OHT + nucleoside (5 μM each) treatments or 3 days of MRT monotherapy, cells were stained using crystal violet, and absorbance was measured at 595 nm using the Cytation 3 Imaging Reader (Agilent). Dose-response curves were fitted by least squares regression with a variable slope (four parameters) using GraphPad Prism version 8.0.0. IC₁₀, IC₂₀, and IC₅₀ values were obtained by interpolating the curves with a 95% confidence interval.

HUVECs were seeded in 24-well plates (2 × 10⁵ cells per well) containing 0.5 mL complete endothelial medium with antibiotics, and were incubated overnight. The next day, 1 plate was fixed and stained to be used as the number of cells at baseline (time = 0 h). For the other plates (time = 24 h to 168 h), the medium was replaced every 72 h with antibiotic-free complete endothelial medium, and the cells were stimulated with IL-6 alone or in combination with sIL-6R. At the end of the incubation periods, the cells were fixed and stained with crystal violet solution. Briefly, the medium was removed and 500 µL of a solution containing 0.1% crystal violet in 20% methanol was added into each well, and the plate was then incubated on a shaker (200 cycles/min) for 20 min at room temperature. Then, the wells were washed 5 times using deionized water and were allowed to air-dry for 24 h. By adding 250 µL of 10% acetic acid solution and shaking the plate for 5 min, the dye retained by the cells was solubilized, and the optical density (OD) in the supernatant was measured at 590 nm using Cytation 3 Imaging reader (BioTek, Winooski, USA). The OD at time = 0 h was set to 1 and a ratio was calculated on the basis of the OD from time = 24 h to 168 h divided by the OD from time = 0 h.