Celltiter glo

Cell viability was evaluated using CellTiter-Glo^® assay (Promega Fitchburg, WI) following the guidelines of the manufacturer. Briefly, at the experimental endpoint, 100 μl of treated cells was transferred into the wells of white, flat-bottomed, opaque 96-well plates (Corning Life Sciences, NY, United States), then 100 μl of CellTiter-Glo^® reagent was added, and the plates were shaken for 2 min and incubated for 20 min at room temperature. Luminescence was recorded using the Spark^® multimode microplate reader (TECAN, Männedorf, Switzerland), with an integration time of 0.1 s per well. Cell viability was evaluated up to 72 h after treatment in dose–response curves, 48 h after treatment in drug combination experiments on AML cell lines, and 24 h after treatment in drug combination experiments on ex vivo primary AML cells.

HepG2 and U87 cells were assessed 24 h posttreatment (NT, 5-ALA, Light, and PDT) by a viability assay based on ATP measurement by bioluminescence (CellTiter-Glo^®, Promega, Madison, WI, USA). A total of 5000 cells of HepG2 or U87 were seeded per well in a 96-well white-walled and clear-bottomed plate (Corning, Amsterdam, The Netherlands) and received 100 μL/well of the CellTiter-Glo mix at room temperature for 10 min, while protected from light. The bioluminescence was then read using a luminometer with MicroWIN software v4.41 (Centro LB960, Berthold Technologies, Bad Wildbad, Germany).

Extracellular ATP was quantified by CellTiter-Glo® assay (Promega). Cells were seeded for 48 h before supernatants were collected for analysis. Fifty microliters of supernatants was added in triplicate to 96-well white tissue culture-treated plates (Corning) and mixed with 50 μL of CellTiter-Glo® reagent. Plates were agitated on a shaker in the dark for 5 min at 37 °C before luminescence was measured using a BioTek Synergy HT plate reader. Results were presented as the mean relative fold change and SD from three independent experiments, with statistical analysis determined by one-way-ANOVA using GraphPad Prism 8.