Gaspak ez anaerobic gas generating pouch system

Trophozoites of G. lamblia (WB strain) were maintained in vitro as described [30 (link)] in TYI-S-33 medium supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL). Cells were harvested during the logarithmic phase of the growth for antiparasitic assessment [31 (link)]. Test compounds were serially diluted and transferred to a white solid bottomed 96-well tissue culture plate (E&K Scientific, Swedesboro, NJ, USA) followed by addition of 5000 G. lamblia trophozoites for a final volume of 100 µL of TYI-S-33 medium and a final concentration of compounds ranging from 50.0 to 0.39 µM. Metronidazole was used as a positive control and assay plates were incubated for 48 h at 37 °C in the GasPak EZ Anaerobic Gas Generating Pouch System (VWR). Parasite viability was measured in triplicate using the CellTiter-Glo luminescence kit in an Envision plate reader [30 (link)]. EC₅₀ values were obtained using the GraphPad Prism 9 Software.

Trophozoites of E. histolytica HM1:IMSS were maintained in vitro as described [29 (link)] in TYI-S-33 medium supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL). Test compounds were serially diluted and transferred to a white solid bottomed 96-well tissue culture plate (E&K Scientific, Swedesboro, NJ, USA) followed by addition of 5000 E. histolytica trophozoites per well for a final volume of 100 µL TYI-S-33 medium and a final concentration of compounds ranging from 50 to 0.39 µM. Metronidazole were used as positive control and assay plates were incubated for 48 h at 37 °C. The plates maintained kept anaerobically using the GasPak EZ Anaerobic Gas Generating Pouch System (VWR). Parasite viability was measured in triplicate using the CellTiter-Glo luminescence kit (Promega G9243) in an Envision plate reader (Perkin Elmer, Waltham, MA, USA) [29 (link)]. EC₅₀ values were obtained using GraphPad Prism 9 Software.