Protocol detail

EPHB4 Inhibitor Treatment of Organoid Cultures

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NCI-H660 cells (ATCC) were seeded at 5000 cells/well density in ultra-low attachment 96-well plates (Corning) and cultured in Hepatocyte growth media (Corning) supplemented with 10 ng/ml epidermal growth factor (Corning), 5% heat inactivated charcoal stripped FBS, 1X Glutamax (Gibco), 5% Matrigel (BD Biosciences), 10 µM ROCK inhibitor (Y-27632, STEMCELL Technologies), and 1X Gentamicin/Amphotericin (Lonza), as described previously^{55 (link)}. At day 8, organoids were treated with NVP-BHG 712 (EPHB4i) from Selleck Chemicals or DMSO (Veh.), and viability was assessed at day 15 as per manufacturer’s protocol (Cell Titer Glo-3D viability assay, Promega).

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Sagar V., Vatapalli R., Lysy B., Pamarthy S., Anker J.F., Rodriguez Y., Han H., Unno K., Stadler W.M., Catalona W.J., Hussain M., Gill P.S, & Abdulkadir S.A. (2019). EPHB4 inhibition activates ER stress to promote immunogenic cell death of prostate cancer cells. Cell Death & Disease, 10(11), 801.

Publication 2019

NCI-H660 cells ultra-low attachment 96-well plates Hepatocyte growth media epidermal growth factor Glutamax Matrigel ROCK inhibitor (Y-27632, Gentamicin/Amphotericin DMSO Cell Titer Glo-3D viability assay

5 heat Amphotericin Assay Cell viability Cells Charcoal Cultured media Dmso Epidermal growth factor Gentamicin Hepatocyte Matrigel Organoids Promega Stemcell Y 27632

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Variable analysis

independent variables

NVP-BHG 712 (EPHB4i) from Selleck Chemicals
DMSO (Veh.)

dependent variables

Viability of NCI-H660 cells

control variables

Cell seeding density at 5000 cells/well in ultra-low attachment 96-well plates
Hepatocyte growth media (Corning) supplemented with 10 ng/ml epidermal growth factor (Corning), 5% heat inactivated charcoal stripped FBS, 1X Glutamax (Gibco), 5% Matrigel (BD Biosciences), 10 µM ROCK inhibitor (Y-27632, STEMCELL Technologies), and 1X Gentamicin/Amphotericin (Lonza)
Treatment duration of 7 days (from day 8 to day 15)

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