96 well black culture plate

ADCC activity of ch2448 was measured using the Promega ADCC Reporter Bioassay (Promega). Briefly, hESCs were harvested using dispase (Stem Cell Technologies) and washed in PBS. Cells were added to a 96‐well black culture plate (Corning) at 30,000 cells per well in mTeSR medium (Stem Cell Technologies). Dilutions of ch2448 were added at 25 μl per well. Engineered effector Jurkat cells, expressing the human FcγIIIA receptor coupled to a downstream gene reporter readout of the nuclear factor of the activated T‐cells (NFAT) ADCC signalling pathway, were thawed and added at approximately 75,000 cells per well. Post 6 hr of incubation, the culture plate was removed and luciferin solution added. Luminescence readout was done using a plate reader (TECAN M2000).

Promastigote forms of L. (L.) infantum (2 × 10⁶ promastigotes/well) were incubated in 96-well black culture plate (Corning Inc., USA) in S10 with the IC₅₀ of triterpenes Lu and UA as well as miltefosine for 0, 10, 20, 30, 40, 50, 60, 120, and 1440 minutes. At each time point, the probes SYTOX Green at 0.5 μM/well (Life Technonologies, USA) or Rhodamine 123 at 3 μM/well (Sigma-Aldrich, USA) were added in the parasite culture in order to evaluate cell membrane damage or mitochondrial membrane potential, respectively. Parasites were placed in the dark for 15 minutes at 25°C, then centrifuged at 1200 g, 5 min at 10°C, and washed three times with 200 μL of PBS. Plates containing parasites stained with SYTOX Green were read in a fluorescence reader using 530 nm emission wavelength and 490 nm excitation, and parasites stained with Rhodamine 123 read with 520 nm emission wavelength and 485 nm excitation. Results were normalized in relation to the control, nontreated parasites. Triton X-100 (0.05 μL) (Sigma-Aldrich, USA) was used as a positive control for cell membrane damage and oligomycin at 0.1 μM/well (Cayman Chemicals, USA) as positive control of mitochondrial membrane potential inhibition.