Infinite f500 luminometer

CHO cells carrying the MEEVS‐reporter‐MAC2 were plated at a density of 2 × 10⁵ in 24‐well tissue culture plates and cultured for 48 h. The conditioned media and cells were separately collected and used to measure luciferase activity. Activities of GLuc and CLuc luciferase secreted into the supernatant were measured using a BioLux® Gaussia Luciferase Flex Assay Kit (NEB) and a BioLux® Cypridina Luciferase Assay Kit (NEB), respectively. Twenty microliter of various conditioned cell culture media were transferred to flat solid bottom and opaque‐walled white 96‐well plates (Nunc, Roskilde, Denmark). GLuc and CLuc bioluminescence values were measured immediately after the addition of 50 μL of substrate solution. Expression of ELuc in cells was measured with the Emerald Luc Luciferase Assay Reagent (TOYOBO). Cells were resuspended at a density of 1 × 10⁵ cells per 50 μL with PBS and transferred to flat solid bottom and opaque‐walled white 96‐well plates (Nunc). ELuc bioluminescence values were measured after the addition of 50 μL of assay reagent and incubation for 10 min. An Infinite F500 luminometer (Tecan, Crailsheim, Germany) was used to determine luciferase activity.

Hydrogen peroxide production was measured in real time using Amplex Red Hydrogen Peroxide/Peroxidase Assay kit (Invitrogen, Waltham, MA), in an Infinite F500 luminometer (Tecan Systems, San Jose, CA). Superoxide was visualized on cell cultures using a treatment with 5 μM 2-hydroxyethidium (DHE, Sigma Aldrich, St. Louis, MO) freshly prepared prior to use from a 10 mM stock solution in DMSO. For that, 25 μl DHE stock solution was diluted in 50 ml Milli-Q pure H₂O. After aspirating most of the media, and 130 ul of DHE staining solution was placed in each culture well and incubated for 20 min at room temperature and away from the light. The cells were washed twice in deionized water for just 1 min. Fluorescence imaging was performed immediately using an Axiovert 200 inverted microscope (Carl Zeiss) with Axio Vision software (version 4.8.1; Carl Zeiss). Fluorescence intensity was calculated in Fiji/ImageJ (National Institute of Health, United States) in tiff files were transformed into 8-bit, and manually thresholded to identify stained cells across all conditions. Measurement values were normalized to the area, in a minimum of six 60x magnification images for each condition in triplicate, and averaged.

The cytotoxicity of PHELA on 293T-ACE.MF cells were, carried out by first seeding 30 000 cells/well/100 µl of the cells in a 96 well plate for 24 h at 37 °C, 5% CO₂ and 95% humidity. After 24 h, a threefold serial dilution of PHELA extracts was performed and 100 µl was transferred to the plate containing cells, except in the cell control wells. The cells were then incubated for 72 h. Following the incubation, the spent media was removed and replaced with fresh 180 µl of growth media and 20 µl of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (5 mg/ml). This was followed by 3 h incubation at 37 °C, the removal of all media, and the addition of 150 µl of 0.1% triton X-Isopropanol. The plate was then incubated for 15 min at room temperature. Afterward, 100 µl of 4 mM hydrochloric acid was added and the plate was read at a wavelength of 620 nm using the Tecan Infinite F500 luminometer.