Figure 6.
OCI-AML-3 CD44KO xenografted cells are less resistant to apoptosis upon treatment with intravenous venetoclax in an intravital imaging zebrafish model. (A) OCI-AML-3CD44KO or its parental cell line was stained with CellTraceViolet (CTV) and injected into the inner cell mass of blastula-stage zebrafish embryos. Upon development, both cell lines were tolerated and engrafted in the CHT. At 2 days after cell implantation, engrafted zebrafish larvae were treated with a single dose of 0.4 nL of venetoclax 2 mM injected into the cardinal vein in combination with a CellEventCaspase3/7 Green Detection Fluorescent Reagent. At 2, 10, and 20 hpi, intravital confocal images were taken and total CTV (blue) cell fluorescence as well as green fluorescence in the CTH was measured using ImageJ. (Bi) Representative grayscale pictures of the 3 time points in the blue channel (CTV) merged with brightfield. (Bii) Plot of the mean total CTV fluorescence in the CHT. (Biii) Plot of the mean total fluorescence of the CHT in the green channel (apoptosis reporter), normalized to the total CTV fluorescence in the same area on each xenografted zebrafish larvae (Student t test; n = 12 engrafted zebrafish larvae per group). Time lapse follow-up of individual engrafted zebrafish larvae of both cell sublines can be seen in supplemental movies. (C) OCI-AML3-EOS-S(4+)d2EGFP cells or OCI-AML3-EOS-S(4+)d2EGFP CD44KO cells were injected into zebrafish embryos as described above and incubated at 33°C. At 3 dpi, the zebrafish larvae were euthanized with ice-cold E3 medium after anesthesia with Tricaine. Larvae tails, which contain the CHT, were microdissected, dissociated by incubation in trypsin-EDTA, strained through a 40-µm mesh, and cultured in normal cell conditions. Upon proliferation, d2EGFP expression was measured by FACS, and the cells were reimplanted into zebrafish embryos initiating the next passage. Representative histograms of 1 experiment and quantification of 3 sequential in vivo passages of 3 independent experiments are shown. The error bars represent standard error. *P < .05; **P < .005. iv, intravenous; PSCs, pluripotent stem cells.

OCI-AML-3 CD44KO xenografted cells are less resistant to apoptosis upon treatment with intravenous venetoclax in an intravital imaging zebrafish model. (A) OCI-AML-3CD44KO or its parental cell line was stained with CellTraceViolet (CTV) and injected into the inner cell mass of blastula-stage zebrafish embryos. Upon development, both cell lines were tolerated and engrafted in the CHT. At 2 days after cell implantation, engrafted zebrafish larvae were treated with a single dose of 0.4 nL of venetoclax 2 mM injected into the cardinal vein in combination with a CellEventCaspase3/7 Green Detection Fluorescent Reagent. At 2, 10, and 20 hpi, intravital confocal images were taken and total CTV (blue) cell fluorescence as well as green fluorescence in the CTH was measured using ImageJ. (Bi) Representative grayscale pictures of the 3 time points in the blue channel (CTV) merged with brightfield. (Bii) Plot of the mean total CTV fluorescence in the CHT. (Biii) Plot of the mean total fluorescence of the CHT in the green channel (apoptosis reporter), normalized to the total CTV fluorescence in the same area on each xenografted zebrafish larvae (Student t test; n = 12 engrafted zebrafish larvae per group). Time lapse follow-up of individual engrafted zebrafish larvae of both cell sublines can be seen in supplemental movies. (C) OCI-AML3-EOS-S(4+)d2EGFP cells or OCI-AML3-EOS-S(4+)d2EGFP CD44KO cells were injected into zebrafish embryos as described above and incubated at 33°C. At 3 dpi, the zebrafish larvae were euthanized with ice-cold E3 medium after anesthesia with Tricaine. Larvae tails, which contain the CHT, were microdissected, dissociated by incubation in trypsin-EDTA, strained through a 40-µm mesh, and cultured in normal cell conditions. Upon proliferation, d2EGFP expression was measured by FACS, and the cells were reimplanted into zebrafish embryos initiating the next passage. Representative histograms of 1 experiment and quantification of 3 sequential in vivo passages of 3 independent experiments are shown. The error bars represent standard error. *P < .05; **P < .005. iv, intravenous; PSCs, pluripotent stem cells.

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