Successful B-cell precursor acute lymphoblastic leukemia (BCP-ALL) graft expansion in zebrafish embryos depends on graft site and host immunosuppression but not on temperature. (A). Viability and growth rate were flow cytometrically assessed for graft cells from groups of 10–20 zebrafish embryos at 35 °C or classical in vitro cultures at 35 °C and 37 °C from three or four independent experiments. (B). Graft cell viability was flow cytometrically assessed 3 days after injection into either the yolk sac (orange) or pericardium (blue). The two independent experiments performed for injections of Nalm-6 into the yolk sac compared to the four experiments with pericardium injections produced a significant difference, with a p-value of 0.007 in an unpaired t-test. Injections were performed in groups of 10–20 embryos. (C). Host embryos were immunosuppressed by morpholino (MO) injection into fertilized eggs (dark blue) and compared to untreated controls (light blue). Graft cell number per host embryo was calculated from flow cytometric analyses from groups of 10–20 embryos in three or four independent experiments. The comparisons of immunosuppressed injections with untreated controls produced p-values in a paired t-test: p = 0.045 for SEM cells, p = 0.095 for Nalm-6 cells and p = 0.049 for RCH-ACV cells. Bars represent means ± SEM. Microscopic images show 5-day old host embryos with (+MO) or without (control) immunosuppression 3 days post-injection (dpi) with DiO-labeled Nalm-6 cell injections into the pericardium. Only one biological replicate was performed for SEM and RCH-ACV injections into the yolk sac. Representative images shown. (D). Representative flowcytometric scatter plots of Nalm-6 cells following engraftment in zebrafish embryos. CD19 positive Nalm-6 cells prelabeled with CellTrace Violet can be separated from auto-fluorescent zebrafish cells to sort out the graft cell population for analysis. Engraftment site indicated as well as whether the host embryo was transiently immunosuppressed using morpholinos (MOs). Groups of 10 embryos from each treatment group were pooled before single-cell dissociation for flow cytometric analysis. Control embryos not engrafted show auto-fluorescence. For details see also Figure S1. Pc = pericardium.