Eclipse ti widefield fluorescence microscope

To measure the development of gradients of intracellular hypoxia of live cells in the REECs, cells were incubated with Image-iT Green Hypoxia Reagent (IGHR) (10 µM; Invitrogen) at 37 °C for 30 min prior to chamber placement. After chamber placement, 4T1 cells were imaged using standard FITC excitation and emission filters at ×4 or ×20 magnification on a Nikon Eclipse Ti widefield fluorescence microscope. To minimize effects of mixing on the live-cell [O₂] measurements, the microscope stage was not moved during data collection, and only a quadrant of the chamber was imaged with the hole in a corner of the image. For controls, IGHR treated 4T1 cells were placed in incubators set to 0.1%, 1%, or 5% O₂ or in a standard incubator (~20%) for 2 h. Cells were immediately imaged (Supplementary Fig. 3).

In order to allow time for disks to form, REECs were placed on a 70–80% confluent monolayer of 4T1-GFP-luc cells, using the method described above, 3 days prior to ANA-1 injection. ANA-1 cells were stimulated with IFNγ and LPS, as described above, 1 day prior to ANA-1 injection. On the day of ANA-1 injection, ANA-1 cells were incubated in serum-free media with CellTracker Red CMTPX Dye (5 µM, ThermoFisher) at 37 °C for 30 min. The ANA-1 cells were then spun down and diluted in complete media (+/−IFNγ and LPS) to ~10,000,000 cells mL⁻¹. Immediately prior to injection, the media in the upper chamber of the REECs was refreshed with complete media (+/−IFNγ and LPS). The concentrated ANA-1 solution (10 µL, containing ~100,000 ANA-1 cells) was injected through the opening, directly into the lower chamber of the REEC. For controls, complete media (10 µL, +/−IFNγ, and LPS) containing no ANA-1 cells was injected. Images were taken every 2 h for 48 h on the Nikon Eclipse Ti widefield fluorescence microscope with a ×4 dry objective and using a live-cell heated stage.

To measure levels of intracellular hypoxia in cell disks, media in the upper chamber were replaced with complete media supplemented with pimonidazole (200 µM; Hypoxyprobe, Inc., Massachusetts) after disk formation. In hypoxic cells, pimonidazole is reduced and forms adducts with thiol groups. After sufficient time for the pimonidazole to diffuse through the chamber and be taken up by cells (~6 h), chambers and media were removed, and the disks were immediately fixed, blocked, and permeabilized as described above. A monoclonal antibody specific to pimonidazole adducts and conjugated with a fluorescein probe (Hypoxyprobe-Green Kit (FITC-Mab); Hypoxyprobe, Inc.) was applied for 1 h at room temperature, or overnight at 4 °C. Samples were rinsed three times with 1× PBS and imaged using standard FITC excitation and emission filters at ×20 magnification on the Nikon Eclipse Ti widefield fluorescence microscope. For controls, cultured 4T1 cells were treated with complete media which had been supplemented with pimonidazole and deoxygenated (<1% O₂) with glucose oxidase and catalase. HIF1α expression was determined by immunofluorescence and correlated with pimonidazole adduct formation (Fig. 1h).