Fig 1.
HIOs exhibit high basal LDH activity.
A) J2, J2 STAT1-/-, J4 and J4 FUT2-/-/FUT2 HIOs were lysed with 0.8% Triton-X 100. Supernatants were diluted 2-fold and used in an LDH reaction using the CytoTox 96® Kit. Non-linear regression. B) HIO monolayers were trypsinized and live cells were counted using trypan blue. One Way ANOVA and post hoc Tukey test. ns = not significant. Data are compiled from n = 2 experiments.
Fig 2.
Modification of the LDH assay yields OD values in the linear range of the assay.
HIOs were lysed with 0.8% Triton-X 100 to release total LDH into the supernatant. A) J2, B) J2 STAT1-/-, and C) J4 samples were used undiluted and diluted 1:20, while D) J4 FUT2-/-/FUT2 samples were used undiluted and diluted 1:10. Reactions were stopped at 5, 10, 20, and 30 minutes and the absorbance at 490 nm was recorded. Data are compiled from n = 3 experiments.
Fig 3.
A cytotoxic antiviral compound demonstrates high variability in estimated CC50 among different HIO lines.
J2, J2 STAT1-/-, J4 and J4 FUT2-/-/FUT2 HIOs were treated with a range of doses with the following antivirals: A) a protease inhibitor (ritonavir), C) DNA polymerase inhibitor (valacyclovir), and E) RNA-dependent RNA polymerase inhibitor (ribavirin). Assays were done 24 h post treatment. 100 minus percent cytotoxicity was measured by the CytoTox 96® Non-Radioactive Cytotoxicity Assay and CellTox™ Green Cytotoxicity Assay. Percent viability was measured by the CellTiter 96® AQueous One Solution Cell Proliferation Assay and the CellTiter-Glo® 2.0 Cell Viability Assay. Viability was validated by quantitation using flow cytometry using propidium iodide and LIVE/DEAD™ dyes. CC50s estimated by non-linear regression for B) ritonavir, D) valacyclovir, and F) ribavirin are plotted. The data from LDH, CellTox, and flow cytometry assays are plotted on the right y-axis (green) and data from the MTS and CellTiter-Glo assays are plotted on the left y-axis (blue). Data are compiled from n = 2 experiments.
Fig 4.
Assay-specific responses in HIOs demonstrate compound-dependent cytotoxicity patterns.
J2, J2 STAT1-/- or J2 FUT2-/-, J4 and J4 FUT2-/-/FUT2 HIOs were treated with the following compounds: A) 80 μM nitazoxanide, B) 100 μM auranofin, C) 100 μM oligomycin A, and D) 60 μg/mL digitonin. Assays were done 24 h post treatment. 100 minus percent cytotoxicity was measured by the CytoTox 96® Non-Radioactive Cytotoxicity Assay and CellTox™ Green Cytotoxicity Assay. Percent viability was measured by the CellTiter 96® AQueous One Solution Cell Proliferation Assay and the CellTiter-Glo® 2.0 Cell Viability Assay. Two Way ANOVA and post hoc Tukey test. Data are compiled from n = 2 experiments.
Table 1.
Summary of benefits and limitations of in vitro cytotoxicity and viability assays used in this study.