Bioassay software

The following inhibitors were purchased: Canavanine and metronidazole (Sigma Aldrich); vincristine, epigallocatechin gallate, and SNX-2112 (Selleck Chemicals); disulfiram (Tocris); and conoidin A (Cayman Chemical). For assays of giardicidal activity, serial 1:3 dilutions of these compounds were made in 96-well plates, G. lamblia WB or GS/M trophozoites were added, and cultures were grown for 1–2 days at 37°C under anaerobic conditions [29 (link)]. Parasite cell growth and viability were determined by measuring ATP levels with the BacTiter-Glo microbial cell viability assay reagent (Promega) in a microplate reader. The 50% effective concentration (EC50) was derived from the concentration-response curves using BioAssay software (Cambridge soft). Acute human cytotoxicity was assayed with the human epithelial cell line, HeLa (ATCC CCL-2) [29 (link),30 (link)]. Compounds were serially diluted (1:3) and added to HeLa cell cultures in 96-well plates. Cells were grown for two days, and viable cell numbers were determined using AlamarBlue reagent (Invitrogen). As done for the EC50 calculations, the 50% cytotoxic concentration (CC50) was derived from the normalized concentration-response curves using BioAssay software (Cambridge soft).

Screening data were analyzed using the CambridgeSoft Bioassay software. The performance of the organoid viability assay in 384-well and 1536-well plates for HTS or uHTS was evaluated by Z’ factor and S/B ratio, which were calculated as the following equations:
$\mathrm{Z}\prime =1 – \left({3\text{SD}}_{\text{DMSO} \text{control}}+{ 3\text{SD}}_{\text{blank}}\right)/\left({\text{FI}}_{\text{DMSO} \text{control}}–{ \text{FI}}_{\text{blank}}\right)$
and
$\mathrm{S}/\mathrm{B}={ \text{FI}}_{\text{DMSO} \text{control}}/{\text{FI}}_{\text{blank},}$
where SD_{DMSO control} and SD_blank are the standard deviations and FI_{DMSO control} and FI_blank are the corresponding average FI signals for the wells with DMSO control and blank with medium only without cells, respectively. A Zʹ factor between 0.5 and 1.0 indicates that the assay is robust for HTS (Zhang et al., 1999 (link)).
The effect of compound on the growth of organoids was expressed as % of Control or % Inhibition based on per plate and calculated as the following equations:
$\% \text{of} \text{Control}=\left({\text{FI}}_{\text{compound}}–{ \text{FI}}_{\text{blank}}\right)/\left({\text{FI}}_{\text{DMSO} \text{control}}–{ \text{FI}}_{\text{blank}}\right)\times 1\mathrm{00}$
and
$\% \text{Inhibition}=1\mathrm{00} – \% \text{of} \text{Control}.$