Visualizing H₂O₂and NO in endothelial cells: strategies and pitfalls

Abstract

The relationship between hydrogen peroxide (H2O2) and nitric oxide (NO) in the vasculature is multifaceted and remains controversial because the dynamic detection of these reactive molecules is challenging. Genetically encoded biosensors (GEBs) allow visualizing real-time dynamics in living cells and permit multiparametric detection of several analytes. Although robust, GEBs' utility depends on several parameters that need fine-tuning for proper imaging and correct data analysis: i.e., camera binning, temperature, and the resolution power of the imaging instruments are some critical parameters that require optimization. We have generated a new double-stable transgenic endothelial cell line stably expressing the biosensors HyPer7 and O-geNOp and systematically tested different imaging modes and their impact on the performance of each biosensor. Ambient temperature and the type of imaging mode did not influence the results, while camera resolution settings significantly affected readouts of HyPer probes but not O-geNOp. Changing a single parameter in a co-imaging mode significantly altered the biosensor's dynamic measurements, potentially causing misinterpretation. This study provides a general guide and the pitfalls of employing GEBs in a multispectral imaging mode.

Genetically Encoded Biosensors Unveil Neuronal Injury Dynamics via Multichromatic ATP and Calcium Imaging

“…The filtered medium was added on top of a 20% sucrose cushion and centrifuged at 10,000 g for 4 h at 4 °C. After centrifugation, viral particles were resuspended in 1× phosphate-buffered saline (PBS) and kept at −80 °C for further usage or the generation of stable HEK293T cells-expressing GRAB ATP1.0 -P2A-MaLionR for coculture experiments following the protocols as described elsewhere . Briefly, HEK293T cells stably expressing GRAB ATP1.0 and MaLionR were selected using a fluorescence-activated cell sorter (FACS), while the top 30% of the cells expressing both biosensors were sorted using 488 nm laser (Filter: 530/40 nm) and 561 nm laser (Filter: 593/40 nm) on a BD Influx Cell Sorter.…”

“…After centrifugation, viral particles were resuspended in 1× phosphate-buffered saline (PBS) and kept at −80 °C for further usage or the generation of stable HEK293T cells-expressing GRAB ATP1.0 -P2A-MaLionR for coculture experiments following the protocols as described elsewhere. 33 Briefly, HEK293T cells stably expressing GRAB ATP1.0 and MaLionR were selected using a fluorescence-activated cell sorter (FACS), while the top 30% of the For the generation of pAAV-GRAB ATP1.0 -P2A-MaLionR/or RCaMP viral particles, 70% confluent AAV293 cells were transfected with pAAV-GRAB ATP1.0 -P2A-MaLionR/or RCaMP, pAdDeltaF6, and pAAV2/1 (gifts from James M. Wilson, Addgene plasmids #112867, #112862) plasmids. The plasmid to PEI transfection reagent ratio was 1:4.…”