Epifluorescence microscopy

Trophozoites of E. histolytica (5×10³ ml⁻¹) were mixed with 5 µl of the active sub-fractions to obtain final concentrations of EC₅₀. The microtiter plate was introduced in Genbag anaer and then incubated at 37°C for 12, 24 and 48 hours and cell viability was evaluated as above described. The same assay was carried out on 8-wells Chamber Slide System (Brumath, France), the chamber was introduced in a Genbag anaer for 12 hours and the morphology of amoebae was examined using indirect immunofluorescence assay. Briefly, amoebae cells were fixed with 500 µl of formaldehyde 3.7% (Thermo scientific, Waltham-MA, USA) for 30 minutes, washed with 500 µl of 3% BSA in PBS and then incubated for 30 minutes at 37°C. The primary antibody Gal/GalNAc lectin (1∶100 diluted in 1% BSA-PBS) was added and the plates incubated for one hour at 37°C in humidified atmosphere. The plates were washed twice with 1% BSA-PBS. The secondary antibody Alexa Fluor 546 goat anti-rabbit IgG (1∶200 diluted in 1% BSA-PBS) was added and incubated for 45 minutes at 37°C. At the end of the experiment, the plates are washed thrice with PBS and the mounting medium Vectashield with nuclear stain DAPI (Vector-ABCYS) was used. For every assay, DMSO is used as negative control. The cells were observed by epifluorescence microscopy (Olympus).

Colon cancer cells and PMA-pretreated THP1 cells were placed on a glass slide and fixed with 5% paraformaldehyde when the cells reached 60–70% confluence. Blocked the cells with 5% donkey serum for 1 h and incubated with the primary antibody overnight at 4 ℃. The antibodies used for the IF assay were listed in Supplementary Table S 4. The next day, the cells were incubated with the corresponding CY3 secondary antibody (Jackson Immunology Research, Erie, UK) at 37℃for 1 h. After counterstaining the nuclei with DAPI (Sigma, St. Louis, Missouri, USA) for 15 min, the fluorescence images were captured by epifluorescence microscopy (Olympus, Tokyo, Japan).(The details are listed in Supplementary Table 4).

Dichlorohydrofluorescein diacetate (DCFHDA) and CellTracker Blue 4-chloromethyl-6,8-difluoro-7-hydroxy-coumarin (CMF₂HC) were used to determine the intracellular levels of ROS and GSH, respectively, as previously described [10 (link),11 (link)] with slight modifications. Briefly, cumulus cells were removed from COCs by pipetting in the presence of 0.1% (w/v) hyaluronidase. Denuded oocytes were incubated in DPBS containing 50 μM DCFHDA or 100 μM CMF₂HC in the dark for 20 min at 38.8°C. Thereafter, oocytes were washed more than five times with DPBS containing 0.1% (w/v) BSA to completely remove excess dye and immediately analyzed by epifluorescence microscopy (Olympus, Tokyo, Japan). The ROS level was measured using excitation and emission wavelengths of 450 to 490 nm and 515 to 565 nm, respectively. The excitation and emission wavelengths of CMF₂HC are 371 and 464 nm, respectively. Grayscale images were acquired with a digital camera (Nikon, Tokyo, Japan) attached to the microscope, and mean grayscale values were calculated using ImageJ software (NIH, Bethesda, MD, USA). Background fluorescence values were subtracted from the final values before statistical analysis. Each experiment was independently repeated 6 to 7 times, with 20 to 30 oocytes per experiment.