Cytation 3 reader

Host cell monolayers in Cell Carrier 96-well plates (PerkinElmer Life Sciences) were infected with 5 × 10⁴ GFP-expressing parasites/well. 18 h postinfection, the medium was exchanged for Ringer's solution, and the intracellular parasites were treated with 10 μm Enh1, 500 μm zaprinast, or 0.5% DMSO. Images were acquired every 10 s for 30 min. To measure the effect of Enh1 on zaprinast and A23187-induced egress, the procedure was repeated, adding either 12.5 μm Enh1 or 0.13% DMSO and imaging for 10 min, before stimulation with 500 μm zaprinast or 1 μm A23187 (Calbiochem) and imaging for an additional 10 min. In all cases, images were acquired with a ×4 objective on a Cytation3 reader (BioTek) using excitation and emission wavelengths of 485 and 528 nm, respectively. Intact vacuoles were defined as objects of at least 78 μm² with a circularity of at least 0.5, as determined in Fiji after default thresholding (41 (link)).

ATP levels were measured using the BacTiter-Glo™ Microbial Cell Viability Assay (Promega). ATP levels were quantified at 24 and 72 h post treatment by adding an equal volume of Bactiter-Glo reagent. Luminescence reading was recorded on a BioTek CYTATION 3 reader.

The metabolic viability of H9c2 cells was assessed by the resazurin reduction assay [45 (link)]. This assay involves the reduction of the blue resazurin dye to a pink fluorescent compound (resorufin) by the respiratory metabolism of the living cells in the medium. The resazurin reduction is a direct measurement of the cellular metabolic activity [48 (link)]. Cells were treated with plant extracts and/or Dox as described in Section 2.6.1. After 24 hours of Dox exposure, the media were removed, and the cells were rinsed with 1% PBS and incubated with 150 µL of culture medium supplemented with 10 µg/mL of resazurin. Reduction to resorufin was measured by fluorimetry at 570 nm excitation and 600 nm emission using a Biotek Cytation 3 reader (Biotek Instruments, Winooski, VT, USA).

Cells were seeded in 96-well plate and then subjected to the different treatments. After incubation time, the cell metabolic activity was assessed through the resazurin reduction assay [36 (link)]. The culture medium was discarded, and cells were incubated for 1 h with 80 μL of culture medium supplemented with 10 μg/mL resazurin. The appearance of resorufin, indicative of metabolic activity, was measured fluorimetrically with 570 nm excitation and 600 nm emission in a Biotek Cytation 3 reader (Biotek Instruments, Winooski, VT, USA).

GSCs were seeded at 20,000 cells per well in white 96-well plates in GSC medium. Cell viability was determined using CellTiter-Glo assay kit (Promega). Each condition consisted of, at least, three replicate wells and then luminescence read using a Cytation3 reader (BioTek). For cell viability assay under hypoxic conditions, GSCs were exposed either to atmospheric O₂ conditions in a conventional hood and incubator (21%), or to 1% O₂ by using the Ruskinn 300 InVivO2 hypoxia workstation (Baker) for 72 h. GSC medium was pre-equilibrated to 1% O₂ by flushing with the corresponding gas mix.

The RAB10/Gal-3 cell-free binding assay was performed as reported^{14 (link)}. Briefly, 96-well plates were coated with purified human Gal-3 (Biolegend, 0.5 µg in 100 µl), incubated at 4 °C overnight, and then blocked with 50 mg/mL bovine serum albumin (BSA) for 90 min at 30 °C. After washing, recombinant human RAB10 (Prospec PRO-1361, from 0.25 to 0.5 µg/well) and MCP were combined and added for a total volume of 100 µl, then incubated for 4 h at 30 °C. Wells were washed, fixed with 2% PFA in PBS for 15 min at room temperature, washed, and then incubated with rabbit monoclonal RAB10 antibody (Abcam, 0.5 mg/mL diluted 1:100) for 1 h at RT. Wells were washed again and incubated with the secondary antibody (Life Technologies A11034, AF488 goat anti-rabbit IgG or A21206, AF488 donkey anti-rabbit IgG, both diluted 1:200) for 1 h at RT. Wells were washed three times and fluorescence read using a Cytation3 reader (BioTek) (ex.: 485 nm, em.: 538 nm) to quantify the binding of RAB10 to Gal-3.