Hypoxia Staining and Analysis in Endothelial Cells

To confirm early and low-dose hypoxia in our endothelial cells, we stained the channel slides with attached HUVEC prior to the hypoxic cycles with Image-iT™ green hypoxia reagent (Invitrogen™) for 20 min according to the manufacturer’s protocol. This fluorescent molecular probe is sensitive to varying concentrations of oxygen, already reacts at O₂ concentrations starting from 5% and due to its irreversible fluorescence is capable of indicating the cumulative hypoxic burden of repetitive hypoxia/normoxia cycles (Zhang et al., 2021 (link)). After 4 h of IH, the slides were disconnected from perfusion systems and were further investigated under the LEICA-TCS SP5 confocal microscope. Images were acquired using the Leica application suite AF software, version 2.7. After microscopy image acquisition, cells were enzymatically detached for a sensitive flow cytometry analysis of the Image-iT™ green hypoxia reagent fluorescence intensity on a single cell level.