Resorufin

The Almar Blue assay was used to assess the metabolic activity of cells. Briefly, 100 µm 7‐hydroxy‐3H‐phenoxazin‐3‐on‐10‐oxid (resazurin; Alfa Aesar, USA) was added to the cells 24 h after treatment, followed by 3 h of incubation under standard culture conditions. Viable cells metabolize nonfluorescent resazurin to fluorescent Resorufin in an NADPH/H⁺‐dependent reaction. Resorufin fluorescence was measured at λ_ex 535nm and λ_em 590 nm using a multimode plate reader (F200; Tecan, Switzerland).

To analyze the metabolic activity of biofilm cells, a modified resazurin assay was established. After cultivation in microtiter plates (see above), biofilms were washed with 200 μl of Brock medium. Then 200 μl of 0.005% (wt/vol) resazurin (sodium salt; Sigma-Aldrich) in Brock medium (pH = 3.0) was added to each of the cavities. In an acidic milieu (pH = 3.0), the oxidoreduction dye resazurin is protonated to resorufin and exhibits a red color. Samples were incubated at 76°C for 3 h. By S. acidocaldarius respiratory activity, resorufin is converted to the colorless product dihydroresorufin. The conversion of resorufin was determined photometrically at 520 nm using a microplate reader (Infinite M200; Tecan). All experiments were performed in triplicates.

The sensitivity of MM cells, leukemia cells, and PBMCs to adapalene was evaluated by the resazurin reduction assay, as previously described [30 (link)]. In brief, 10⁴ cells were seeded in each well of a flat bottom 96-well plate. Cells were directly treated with 10 different concentrations of adapalene (Activate Scientific, Prien am Chiemsee, Bavaria) which are 3-fold apart from one another, ranging from 100 µM to 0.003 µM for MM and T-ALL cells. However, when 10-fold apart from one another, they ranged from 100 µM to 10⁻⁷ µM for PBMCs. The plates were incubated for 72 h at 5% CO₂/37 °C and then re-incubated for another 4 h under the same conditions, with 20 μL of resazurin (0.01% w/v; Sigma-Aldrich) added to each well. An Infinite M2000 Pro plate reader (Tecan, Crailsheim, Germany) was used to measure resorufin fluorescence, produced by the reduction of resazurin by live cells, at 544–590 nm (excitation-emission wavelengths). Afterward, cell viability was plotted vs. adapalene concentration, and the IC₅₀ values from three separate experiments with six repeats each were calculated with GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA).

To analyze metabolic activity, 50 µL of fully supplemented cell culture medium containing resazurin (Sigma, Taufkirchen, Germany) at a final concentration of 100 µM was added to the cells. resazurin freely diffuses into cells, where it is reduced by nicotinamide adenine dinucleotide phosphate (NADPH) to its fluorescent product, resorufin. After 4 h of incubation, the plate was transferred to an F200 microplate reader (Tecan, Männdedorf, Switzerland), and resorufin fluorescence was quantified at λ_ex 535 nm and λ_em 590 nm. For each cell line, the relative fluorescence units obtained were normalized to the respective untreated controls.

After receiving the different substances from the kinase-inhibitor library (+/− H₂O₂), the cells were stored at optimal growing conditions for 24 h hours before resazurin (Alfa Aesar, Haverhill, MA, USA) was added at a final concentration of 100 µM. This metabolite can be converted by viable active cells to the fluorescent resorufin. The fluorescence of resorufin was then quantified using a multiplate reader (Tecan, Männedorf, Switzerland) at λ_ex 560 nm and λ_em 590 nm. Normalization was performed depending on the experimental question to either the untreated control or H₂O₂ alone.

Resazurin (Sigma) was added to the cell culture medium (5 μg/ml) for a period of at least 30 min. Resorufin fluorescence was determined at 530 nm_ex/590 nm_em with a Tecan Infinite M200 reader. Cell viability was expressed as percentage of fluorescence intensity relative to untreated controls. Background fluorescence was determined in each individual experiment with cells exposed to Triton (1%) and subtracted from all other values.