Dtx 880 multimode plate reader

Cells were seeded in 96-well white plates 24 h prior as follows: Vero and Caco-2 cells at 5 × 10⁴ cells per well, Calu-3 cells at 1.3 × 10⁵ cells per well. Inhibitors were diluted to the desired micromolar concentration (μM) in the appropriate culture media. Diluted compounds were added to the cells and plates were incubated at 37 °C, 5% CO₂. At 24 h post-treatment (hpt), culture media were removed from the cells and cell viability was measured using a CellTiter-Glo^® Luminescent Cell Viability Assay per manufacturer’s instructions (Promega, G7572, Madison, WI, USA). Luminescence was measured using a Beckman Coulter DTX 880 Multimode plate reader with Multimode Analysis Software Version 3.3.0.9.

We performed ELISA on conditioned media after 48 hours to assess the production of eotaxin-3 by esophageal epithelial cells. Conditioned media from the cells were collected and centrifuged to remove cellular debris. Eotaxin-3 concentrations were determined using commercially-available ELISA kits (R&D Systems) per manufacturer's instructions. The absorbance of each well was read at 450 nm and 540 nm using a DTX 880 Multimode plate reader (Beckman Coulter). Results were expressed as pg/ml of eotaxin-3. All assays were performed in duplicate.

At indicated time points, supernatants were collected or cellular lysates were obtained using 1X Passive Lysis Buffer (E1941, Promega, Madison, WI, USA) and the Nano-Glo Luciferase Assay System (N1130, Promega) was used to measure luciferase activity per the manufacturer’s instructions. An aliquot of cellular lysates were mixed with Bradford Reagent (5000006, Bio-Rad, Hercules, CA, USA) per the manufacturer’s instructions. A standard curve for total protein was established using Bovine Serum Albumin (BSA, BP1600, FisherSci) diluted in Passive Lysis Buffer at concentrations of 1, 2.5, 5, 10, 15, 20, 25 µg/µL. Mock-infected cells were used to establish the limit of detection for luciferase assays. Luminescence and absorbance was measured using a DTX 880 multimode plate reader (Beckman Coulter, Brea, CA, USA). Intracellular luciferase was normalized to total µg of protein. Overall, sensitivity of the DTX 880 multimode plate reader was somewhat limited due to the age and performance of the machine with lower than anticipated RLU values observed following infection.

We performed ELISA of conditioned media to assess the secretion of eotaxin-3 by fibroblasts. Conditioned media collected were centrifuged to remove cellular debris. Cytokine concentrations were determined using commercially available ELISA kits (R&D Systems) per manufacturer’s instructions. The absorbance of each well was read at 450 nm and 540 nm using a DTX 880 Multimode plate reader (Beckman Coulter, Brea, CA).

Nitric oxide production was determined by Griess assay to detect ${\text{NO}}_2^{-}$ in cell culture supernatants. Equal volumes of cell culture supernatant and Griess reagent (0.5 g sulfanilamide (S9251, Sigma-Aldrich), 0.05 g N-(1-Naphthyl)ethylenediamine dihydrochloride (33461, Sigma-Aldrich), and 1.17 mL phosphoric acid per 50 mL water) were mixed in a 96-well plate. Sodium nitrite (237213, Sigma-Aldrich) was used as a standard. Absorbance values were measured immediately at 492 nm using a DTX-880 Multimode plate reader and detection software (Beckman Coulter).

Macrophage proliferation was determined by BrdU incorporation (6813, Cell Signaling) according to the manufacturer’s protocol. BrdU was detected following 72 hours of incorporation (PMΦs) or 20 hours of incorporation (RAW 264.7). Absorbance values at 450 nm were obtained using a DTX-880 Multimode plate reader (Beckman Coulter) or Synergy Neo2 Multi-Mode Reader and Gen5 software (BioTek).