Sparks et al, Heterogeneity in tumor chromatin-doxorubicin binding revealed by in vivo fluorescence lifetime imaging confocal endomicroscopy: In vitro data

Abstract

Cell lines

IGROV-1 cell lines were cultured in CO2 dependent media with 10% fetal bovine serum and 1% Pen Strep at 37 ˚C. Before experiments, cells were grown to 80% confluence. For measuring doxorubicin uptake by fluorescence an IGROV-1 cell line stably expressing GFP fused to Histone-1 (H1) was made using the PiggyBac transposon system. As a control to show that effect of doxorubicin on GFP depends on whether it is fused to H1 or not, a stable whole cell expression of GFP by lentiviral transfection and selection by Geneticin was made. For bioluminescence imaging of xenograft tumors, all IGROV-1 cell lines were made to stably express firefly luciferase.

To investigate the effect of doxorubicin on other histones, IGROV-1 cells were transiently transfected with a Histone-2B-GFP plasmid (gift from Kurt Anderson) using the Lipofectamine® 2000 reagent.

IGROV-1 cells were obtained from Crick institute cell services and confirmed as IGROV-1 by Short Tandem Repeats (STR) profiling and no mycoplasma was detected.

In vitro experiments

IGROV-1 cells were grown to 80% confluence in 75 ml flasks before being re-plated in 12 or 24 well plates or 35 ml glass bottomed dishes and allowed to attach to the surface for 24 hours before experiments.

To study how the fluorescence of GFP labelled H1 labelled IGROV-1 cells changes with doxorubicin treatment, fluorescence intensity and lifetime distributions were measured from cells after 3 hours of incubation with doxorubicin of varying concentrations (0, 0.18, 0.9, 1.8, 9, 18 µM) by serial dilutions of a stock solution with PBS. After 3 of hours, cells were washed in PBS then fixed for 20 minutes in 4% PFA. Cells were then imaged in PBS. Doxorubicin hydrochloride (Sigma-Aldrich, D1515-10 mg) was dissolved in PBS to a concentration of 9 mM and stored at -20˚C.

Cell lines IGROV-1 cell lines were cultured in CO2 dependent media with 10% fetal bovine serum and 1% Pen Strep at 37 ˚C. Before experiments, cells were grown to 80% confluence. For measuring doxorubicin uptake by fluorescence an IGROV-1 cell line stably expressing GFP fused to Histone-1 (H1) was made using the PiggyBac transposon system. As a control to show that effect of doxorubicin on GFP depends on whether it is fused to H1 or not, a stable whole cell expression of GFP by lentiviral transfection and selection by Geneticin was made. For bioluminescence imaging of xenograft tumors, all IGROV-1 cell lines were made to stably express firefly luciferase. To investigate the effect of doxorubicin on other histones, IGROV-1 cells were transiently transfected with a Histone-2B-GFP plasmid (gift from Kurt Anderson) using the Lipofectamine® 2000 reagent. IGROV-1 cells were obtained from Crick institute cell services and confirmed as IGROV-1 by Short Tandem Repeats (STR) profiling and no mycoplasma was detected. In vitro experiments IGROV-1 cells were grown to 80% confluence in 75 ml flasks before being re-plated in 12 or 24 well plates or 35 ml glass bottomed dishes and allowed to attach to the surface for 24 hours before experiments. To study how the fluorescence of GFP labelled H1 labelled IGROV-1 cells changes with doxorubicin treatment, fluorescence intensity and lifetime distributions were measured from cells after 3 hours of incubation with doxorubicin of varying concentrations (0, 0.18, 0.9, 1.8, 9, 18 µM) by serial dilutions of a stock solution with PBS. After 3 of hours, cells were washed in PBS then fixed for 20 minutes in 4% PFA. Cells were then imaged in PBS. Doxorubicin hydrochloride (Sigma-Aldrich, D1515-10 mg) was dissolved in PBS to a concentration of 9 mM and stored at -20˚C.