Wallac 1420 victor plate reader

Each well of a 96-well culture plate was seeded with 4000 cells of; NIH/3T3, A-375 and LLC, in 100 μl media (DMEM, 10% FCS) and incubated in 37°C for 24 hours. A total of three plates with each cell line seeded in a separate plate. Treatment of Carbenoxolone in varying concentrations (0, 0.1, 0.5, 1, 5 and 10 μM) was added in another 100 μl media and cells were incubated in 37°C for another 72 hours. After the incubation, WST-8 reagent was added into each well and cells were incubated in 37°C for 1 hour. Absorbance was measured at 450 nm using a plate reader (Wallac 1420 VICTOR plate-reader, Perkin-Elmer Life Sciences, USA).

AsPC-1 cells were seeded on fabricated polyacrylamide gels or on commercial gels at 300 000 cells per well with their complete culture medium. Cells were allowed to adhere and grow for 12 or 96 h. After incubation, MTT (3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent was added into each well for metabolic activity detection and incubated at 37 °C with 5% CO₂ for 4 h. Absorbance was measured at 540 nm using a plate reader (Wallac 1420 VICTOR plate-reader, Perkin-Elmer Life Sciences, USA). Absorbance values were transformed to metabolic activities, and percentage was calculated as the fraction of control cells that were cultured on standard 6-well plates (fixed as 100% metabolic activity).
Apoptosis was measured by staining cells with annexin V-APC and propidium iodide (PI) (Biolegends, San Diego, California, USA) according to the manufacturer's instructions after cells were grown on commercial gels for 12 h. After 12 h, cells were collected by centrifugation and washed with PBS. Ten thousand events per sample were recorded by FACS. Data analysis was performed using Flow-Jo software (BD Bioscience, USA).

For the cytotoxicity assay, the cells were seeded into a 96-well culture plate at a density of 5 × 10³ cells per well. MTT-assay was performed similarly as described recently [31 (link)]. Briefly, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) conversion was used to evaluate the metabolic activity of the cells after their exposure to antibiotics, according to the manufacturer’s instructions. Absorbance was measured using Wallac 1420 VICTOR plate reader (Perkin Elmer, Waltham, MA, USA). The mitochondrial activity of the cells was measured after 24 h, 48 h and 72 h of exposure.

The effect of ECM on Carbenoxolone-mediated cell adhesion, was studied using five different coatings with ECM component. Each well of a 24-well culture plate was coated with 0.5 ml of: elastin, fibronectin, collagen (I), gelatin and laminin (50 μg/ml) and one plate was left uncoated for control. Coatings were left 2 hours at RT. Plates were washed with 1 ml PBS and incubated with media for 30 minutes in 37°C. LLC cells were harvested and counted. 2 × 10⁶ cells were suspended in 2 ml PBS, 10 μl DiO were added with the tube kept on ice under aluminum foil for 20 min. 6.7 × 10⁴ were seeded in each well and incubated in 37°C, under treatment of Carbenoxolone for 1 hour. Cells were washed twice with PBS and total fluorescent signal was measured (at the central field) using a plate reader (Wallac 1420 VICTOR plate-reader, Perkin-Elmer Life Sciences, USA) at Ex/Em of 480/530.

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA) was used to evaluate the viability of the LESC after exposure to LPS to determine the optimal concentration for LESC stimulation^{27 (link)}. Absorbance referring to the concentration of formazan was measured using Wallac 1420 VICTOR plate reader (Perkin Elmer, USA) after 24 h of exposure to 0.1 µg/ml; 0.2 µg/ml; 0.5 µg/ml; 0.7 µg/ml; 1 µg/ml and 2 µg/ml of LPS and compared to control. All groups were analyzed in six replicates.

The number of viable cells was determined by the mitochondrial conversion of yellow MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan dye. MTT (Sigma) was dissolved in Hank’s balanced salt solution (HBSS; Sigma), filtered, protected from light, and stored at 4 °C. HAPI cells were seeded on 96-well plates at a density of 15,000 cells per well. The next day, cells were treated with various concentrations of P. mirifica extract or LPS (100 ng/mL) and incubated for 24 h. The medium was removed and 10 μL of 10 mg/mL MTT in HBSS was added to each well. The cultures were incubated for 4 h in a humidified atmosphere at 37 °C and 5% CO₂. Then, MTT was removed, cells were solubilized with 100 μL dimethyl sulfoxide, and the absorbance was measured at 570 nm on a microplate reader (Wallac 1420 Victor plate reader, Perkin-Elmer, Foster City, CA, USA). The results are shown as the percentage of the control (no treatment).

Carbenoxolone was previously shown to increase the susceptibility of cells through the induction of apoptosis under non-adherent conditions, i.e., anoikis using poly(2-hydroxyethyl methacrylate) (p-HEMA) coated plates (Sigma-Aldrich, Cat. No. P3932). To evaluate the activity of drug-loaded particles, the same bioassay was applied. Briefly, a solution containing 20 mg/mL p-HEMA in 95% ethanol was prepared and left at room temperature to dissolve for 48 h. Once dissolved, the solution was pipetted into 6-well culture plates. The plates were left half covered in a sterile environment on a rocking plate until the ethanol evaporated and the p-HEMA solidified, coating the plates evenly. The plates were then washed twice with PBS to remove any possible traces of ethanol. Each plate was incubated with growth media containing either empty or carbenoxolone-loaded particles at a concentration of 100 µg/mL. All the plates were seeded with 50,000 LLC cells/well and incubated for 72 h at 37 °C. Cell viability was measured using WST-8 (Sigma-Aldrich, Cat. No. 96992) according to the manufacturer’s protocol, and the cells were incubated at 37 °C for 90 min. Absorbance was measured at 450 nm using a plate reader (Wallac 1420 VICTOR plate-reader, Perkin-Elmer Life Sciences, Waltham, MA, USA) and bright-field images were obtained using an Olympus IX-73 microscope.