Formaldehyde 3.7

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).

The following reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA): B-27^® supplement 50x, Fluo-4 AM cell permeant, formaldehyde 37%, GlutaMAX™ supplement, human recombinant epidermal growth factor (EGF), MEM non-essential amino acids solution 100x, N-2 supplement 100x, and phosphate buffered saline 1x (PBS). The following reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA): 2,5-hexanedione, γ-Aminobutyric acid (GABA), bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), Dulbecco’s Modified Eagle Medium (DMEM), endosulfan, methylmercury, penicillin-streptomycin solution 100x, Triton™ X-100, and TWEEN^® 20. Matrigel^® was purchased from Corning (Corning, NY, USA). Fetal calf serum (FCS) was purchased from ATCC (Manassas, VA, USA). Human recombinant fibroblast growth factor basic (bFGF) was purchased from Peprotech (Rocky Hill, NJ, USA). Tetrodotoxin (TTX) was purchased from Tocris Bioscience (Bristol, UK).