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

<p>Data is divided into three folders:</p> <ul> <li>Sparks_et_al_FIG2_Histone_vs_free_GFP <ul> <li>data for Sparks et al Figure 2</li> <li>main text section: <em>&#39;FRET between &nbsp;chromatin-bound GFP and doxorubicin&#39;</em></li> </ul> </li> <li>Sparks_et_al_FIG3_in_vitro_dose_response <ul> <li>data for Sparks et al Figure 3#</li> <li>main text section:<em> &#39;FLIM endomicroscope can monitor doxorubicin cellular uptake&#39;</em></li> </ul> </li> <li>Sparks_et_al_SuppFIG2_endoscope_spectral_cross_talk <ul> <li>data for Sparks et al Supplementary Figure 2</li> <li>Supplementary information</li> </ul> </li> </ul> <p><strong>Cell lines</strong></p> <p>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.</p> <p>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&reg; 2000 reagent.</p> <p>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.</p> <p><strong>In vitro experiments</strong></p> <p>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.</p> <p>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 &micro;M) 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.&nbsp; Doxorubicin hydrochloride (Sigma-Aldrich, D1515-10 mg) was dissolved in PBS to a concentration of 9&nbsp;mM and stored at -20˚C.</p>