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

Title: Sparks et al, Heterogeneity in tumor chromatin-doxorubicin binding revealed by in vivo fluorescence lifetime imaging confocal endomicroscopy: In vitro data
Authors: Sparks, H
Kondo, H
Hooper, S
Item Type: Dataset
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.
Issue Date: 17-May-2018
Citation: 10.1038/s41467-018-04820-6
URI: http://hdl.handle.net/10044/1/64496
DOI: https://doi.org/10.5281/zenodo.1249014
Copyright Statement: https://creativecommons.org/licenses/by/4.0/
Keywords: tumor chromatin-doxorubicin binding
in vivo fluorescence
confocal endomicroscopy
In vitro data
Notes: Data is divided into three folders: Sparks_et_al_FIG2_Histone_vs_free_GFP data for Sparks et al Figure 2 main text section: 'FRET between chromatin-bound GFP and doxorubicin' Sparks_et_al_FIG3_in_vitro_dose_response data for Sparks et al Figure 3# main text section: 'FLIM endomicroscope can monitor doxorubicin cellular uptake' Sparks_et_al_SuppFIG2_endoscope_spectral_cross_talk data for Sparks et al Supplementary Figure 2 Supplementary information
Appears in Collections:Faculty of Natural Sciences - Research Data



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