Zebrafish Xenotransplant Cancer Model for Drug Screening - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 216

however, horseradish peroxidase (HRP)-conjugated secondary antibody (Bio-Rad,
Hercules, CA) replaced Alexa Fluor 488-conjugated antibody. Peroxidase suppressor
was used to inhibit endogenous HRP and Triton-100 treatment was used to increase
antibody permeability. Xt zebrafish without primary antibody staining served as a
control. After extensive washing, zebrafish were distributed into the wells of
transparent, black, flat-bottom microplates (Corning Life Sciences, Lowell, MA)
and PS-atto (Beckman Coulter/Lumigen Inc., Southfield, MI) was used as the
enzyme substrate that was quantified in Xt cells by measuring chemiluminescence
intensity of the end product using a Bio-Tek ELX-800 microplate reader (Bio-Tek,
Winooski, VT).

17.3.8 Establish Assay Robustness and
Reproducibility

In order to avoid skewing results in large-scale drug screening, it is essential to initially
establish robustness and reproducibility of signal ( S ) generated in the biological process
in the absence of test compounds, followed by compound screening to establish assay
and screen quality. To optimize assay quality, the end signal must be consistent within
and between plates and control compounds must exhibit expected activity. A general
layout for assessing plate reproducibility is illustrated in Fig. 17.1, where H represents
the highest signal, M represents medium signal, and L represents the lowest signal. H,
M, and L were assessed in each position on the plate. This study was performed twice
per day on two different days. We used 1% DMSO-treated Xt zebrafish as H because
chemiluminescence signal was highest, 1000 mM 5-FU-treated Xt zebrafish as M
because signal was in the middle of the range, and the Xt zebrafish without primary
antibody incubation as L because signal was lowest.
Based on results of this study for assessing plate reproducibility, we determined
mean, SE, and CV (%) for each signal (H, M, and L) on each plate. We determined
signal-to-noise ratio (background measurement, S/N); S/N ratio 2.3 is considered

acceptable. We then calculated the Z -factor following formula 17.1. An assay with

Figure 17.1 Microplate layout for assessing reproducibility. H, maximum signal sample; M,
medium signal sample; L, minimum signal sample. One percent DMSO-treated Xt zebrafish, H;
1000

m M 5-FU-treated Xt zebrafish, M; Xt zebrafish without primary antibody incubation, L.