A Co-Culture-Based Multiparametric Imaging Technique to Dissect Local H2O2 Signals with Targeted HyPer7

Abstract

Multispectral live-cell imaging is an informative approach that permits detecting biological processes simultaneously in the spatial and temporal domain by exploiting spectrally distinct biosensors. However, the combination of fluorescent biosensors with distinct spectral properties such as different sensitivities, and dynamic ranges can undermine accurate co-imaging of the same analyte in different subcellular locales. We advanced a single-color multiparametric imaging method, which allows simultaneous detection of hydrogen peroxide (H2O2) in multiple cell locales (nucleus, cytosol, mitochondria) using the H2O2 biosensor HyPer7. Co-culturing of endothelial cells stably expressing differentially targeted HyPer7 biosensors paved the way for co-imaging compartmentalized H2O2 signals simultaneously in neighboring cells in a single experimental setup. We termed this approach COMPARE IT, which is an acronym for co-culture-based multiparametric imaging technique. Employing this approach, we detected lower H2O2 levels in mitochondria of endothelial cells compared to the cell nucleus and cytosol under basal conditions. Upon administering exogenous H2O2, the cytosolic and nuclear-targeted probes displayed similarly slow and moderate HyPer7 responses, whereas the mitochondria-targeted HyPer7 signal plateaued faster and reached higher amplitudes. Our results indicate striking differences in mitochondrial H2O2 accumulation of endothelial cells. Here, we present the method’s potential as a practicable and informative multiparametric live-cell imaging technique.

Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels

“…4 ). However, treatment of HEK293T and EA.hy926 cells with 1 mM FeSO 4 and 1 mM ascorbate led to robust increases in mitochondrial H 2 O 2 levels and significant decreases in the cell cytosol measured using the ultrasensitive H 2 O 2 biosensor HyPer7 [ 35 , 45 ] ( Fig. 4 c and d).…”

“…A cytosol-targeted O-geNOps (O-geNOp-NES) construct was subcloned into a 3 rd generation lentivirus shuttle vector pLenti-MP2 (Addgene #36097) using the following primers: forward 5’-ATAGGATCCGCCACCATGGTGAGTGTG-3' including BamHI restriction site and reverse 5’-ATAGTCGACTTACAAAGTCAATCTTTCT-3' including a stop codon and SalI restriction site. Stable EA.hy926 cell line generation was achieved following optimized protocols as recently described [ 35 ]. After positive transduction, cells were cultured for one week in fresh complete DMEM before fluorescence activated cell sorting (FACS).…”

“…Real-time cell imaging experiments were performed on inverted widefield epifluorescent microscopes, either an Axio Observer.Z1/7 or Axio Vert.A1 (Zeiss, Germany), equipped with a Plan-Apochromat 20×/0.8 dry objective, a Plan-Apochromat 40×/1.4 DIC (UV) VIR-IR oil immersion objective, and monochrome CCD cameras Axiocam 503. O-geNOp and HyPer7 expressing cells were imaged as described elsewhere [ 26 , 35 , 36 ].…”

“…For experiments under physiological normoxia, cells were cultured in O 2 -regulated workstations (Scitive or InVivo 500, Baker-Ruskinn, U.S.A.) for at least 5 days with O 2 maintained at 5 kPa O 2 , CO 2 at 5% and 37 °C [ 31 , 32 , 40 , 41 ]. This experimental protocol enable adaptation of the cell redox proteome to a defined pericellular O 2 level avoiding re-exposure of adapted cells to room air during trypsinization and/or treatments [ 35 , 38 ] Moreover, as shown in Supplementary Fig. 1 , adaptation of cells to 5 kPa O 2 for 5 days avoids stabilization of HIF-1 associated with acute decreases in ambient O 2 levels [ 40 , 41 ].…”

Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors

“…Stable EA.hy926 ] and HEK293T cell lines expressing O-geNOps-NES [ 24 ] used in this study were developed in previous studies [ 25 ]. The same protocols were applied to generate stable HeLa cells, as described previously [ 29 ]. Cells with Mito-C-geNOps, stably O-geNOps-NES expressing HeLa, were seeded on 30 mm glass coverslips for transient transfection.…”