Application suite af software version 2

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.

MitoTracker Green FM (#9074; Cell Signaling Technology, Danvers, MA, USA) was used to determine the mitochondrial mass via flow cytometry analysis. Cells were incubated with 200 nM MitoTracker in the dark at 37°C for a duration of 15 min. Mitochondrial mass per cell was subsequently determined by quantification of mean fluorescence intensity (MFI) green using a FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
For microscopic imaging, CD4⁺ and CD8⁺ T cells were incubated as described above. After two additional washing steps, lymphocytes were seeded in µ‐slides 2 × 9 well (Ibidi, Gräfelfing, Germany). Images of 10 randomly chosen microscopic fields per well at 200× magnification and 3× digital zoom were acquired on a LEICA‐TCS SP5 Confocal Microscope (Leica, Wetzlar, Germany) using Leica application suite AF software, version 2.7.