Resazurin

For both treatment strategies, the viability of the GC-1 cells was measured using the Resazurin reduction assay. Viable cells with an active metabolism contain coenzymes, such as NADH, which are used in diaphorases to reduce Resazurin (blue, non-fluorescent) to resorufin (pink, fluorescent) [37 (link)]. The level of reduction is proportional to the number of viable cells. Resazurin was chosen to perform a cell viability assay because it is not toxic and, therefore, cells exposed to it can remain in the culture to be used for other experimental purposes [38 (link)].
The cells were incubated with 10% of the culture volume with a stock solution of Resazurin (Sigma-Aldrich) (0.1 mg/mL) in 1x PBS [39 (link)] 4 h before the end of the 6 and 12 h incubation timepoints and 4 h before the end of the 4-day recovery period. After 4 h of incubation, 100 µL of the supernatant from each sample was transferred to a 96-well plate, and the absorbance of Resazurin was measured spectrophotometrically at 570 and 600 nm (Infinite M200, PRO, Tecan). All absorbance values were corrected against blank wells containing cell-free culture medium with ZnO NPs and different concentrations of chalcone 1. Cells were visualized daily, under an inverted light microscope (EVOS™ M5000 Imaging System), to confirm cell confluence and morphology.

The antiproliferative
activity of the 15 compounds was evaluated using the resazurin assay.
CEM-CCRF cells were first exposed to the compounds at a fixed screening
concentration (10 μM). To determine the IC₅₀ values of the selected most active compounds, 10 different concentrations
in the range 0.3–100 μM were used for each of compound.
Both CEM-CCRF and CEM/ADR5000 suspension cells were treated immediately
after seeding. After 72 h incubation, 20 μL 0.01% resazurin
(Promega, Mannheim, Germany) was added to each well. resazurin fluorescence
was measured after 4 h incubation using an Infinite M2000 Pro plate
reader (Tecan, Crailsheim, Germany) at Ex/Em = 550 nm/590 nm wavelength.^{44 (link),45 (link)} Cell viability was calculated in comparison to DMSO employed as
the negative control. The final concentration of DMSO in the assay
medium was 0.5%. The anticancer drug cisplatin was used as the positive
control. This experiment was performed in triplicate with six wells
each for each concentration.

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).

Cell viability was further tested using fluorometric analysis by Resazurin (Invitrogen) assay. Resazurin was diluted in cell-growth media in a concentration of c = 560 µM. 200 µl solution was added on the cultured cells (incubated with the investigated material) in 96-well plates and left incubating for 4 h. After 4 h the solution was removed from the wells and tested on a fluorescence microplate reader (Tecan, Infinite M1000). Resazurin is irreversibly reduced to the pink-colored and highly red fluorescent resorufin during oxidation-reduction processes in cell mitochondria, indicating the mitochondrial function and thus cell viability. The emission fluorescence spectrum of resorufin was recorded using λ = 560 nm excitation light. The spectral peak intensity at approximately λ = 560 nm directly indicates the cell viability.

The proliferation inhibition activity of the four compounds was detected using the resazurin assay. CEM-CCRF were first exposed to the test compounds at a fixed concentration of 10 μM. For IC₅₀ determination of the selected compounds, 10 concentrations were prepared for each of the compounds in a range of 0.3–100 μM. Both CEM-CCRF and CEM/ADR5000 suspension cells were treated immediately after seeding. After 72 h incubation, 20 μL of 0.01 % resazurin (Promega, Mannheim, Germany) was added to each well. resazurin fluorescence was measured after 4 h incubation using an Infinite M2000 Pro plate reader (Tecan, Crailsheim, Germany) at Ex/Em = 550 nm/590 nm wavelength [41 (link),42 (link)]. Cell viability was calculated in comparison to DMSO control. The final concentration of DMSO was 0.5 %. Cisplatin was used as positive control. This experiment was repeated three times with one of the six wells for each concentration. Cell viability was calculated in comparison to DMSO control.

Cells were seeded in 96-well plate (8 × 10³ cells/well for fibroblasts and 5 × 10³ cells/well for PDAC cells), and after 24 h they were treated with the various compounds at the concentrations and time points indicated in figure legends. At the end of the treatment period, cell proliferation for parental adherent PDAC cell lines was measured by crystal violet assay in accordance with the protocol of the manufacturer, and absorbance (A_595nm) was measured by spectrophotometric analysis (GENios Pro, Tecan, Milan, Italy). Three independent experiments were performed for each assay condition.
Cell proliferation for PDAC CSCs growing in suspension was evaluated using Resazurin assay (Immunological Science, Rome, Italy), which is based on the reduction of oxidized non-fluorescent blue Resazurin to a red fluorescent dye (resorufin) by the mitochondrial respiratory chain in live cells. Resazurin solution was added in each well, and after 1 h, fluorescence was measured by Tecan GENios Pro Microplate reader (Ex₅₃₅nm, Em_590nm). The quantity of resorufin produced is directly proportional to the amount of living cells. Three independent experiments were performed for each assay condition.

Reduction of the redox dye resazurin to resorufin was used to measure the proliferation of cell cultures.^{17 (link),18 (link)} Briefly, chondrocyte cells were seeded at 1 × 10⁵ of trypan blue excluding cells/mL in 24 or 96-well microtitre plates. After 24 h, cells were exposed to various concentrations of vancomycin (1.25, 2.5, 5 and 10 mg/mL) for a further 24 h. Following the appropriate incubation, the culture medium was removed and replaced with fresh medium containing 44 μM of resazurin (Sigma-Aldrich, St Louis). Cultures were then incubated for a further 1 h and subsequently the reduction of resazurin to resorufin was determined using fluorescence (excitation 530 nm; emission 590 nm) using Tecan Infinite M200 Pro (Tecan, Mannedorf, Switzerland).