Fluospheres carboxylate modified microsphere

In vivo ciliary flow recordings were performed on P10 mouse ventricles. Lateral ventricles were isolated and immediately dissected in cold DMEM supplemented with 25 mM HEPES, followed by sectioning using a Leica VT1000S vibratome (Leica, Wetzlar, Germany) to a final thickness of 500 μm. Sections were maintained in DMEM containing 25 mM HEPES at RT during the recording period. FluoSpheres Carboxylate-Modified microspheres (0.5 μm yellow-green; F8813, Thermo-Fisher, Waltham, MA) were used to track the flow generated by ependymal cilia. Live images were recorded at 300 frames per second using an Ultima In Vivo multiphoton laser scanning microscope (Bruker Nano Fluorescence Microscopy, Billerica, MA) equipped with a 10X immersion objective and an optical zoom of 80X. Video rate image acquisition was achieved by using the Ultima system resonant scanners. Imaris software was used to analyze the tracked fluorescent particles and calculate the average speed of the beads (Bitplane AG, Zurich, Switzerland).

To assess perfusability of the microvascular networks, 2 µm yellow-green fluorescent microbeads (FluoSpheres Carboxylate-Modified Microspheres, 2% solids, Thermo Fisher) were introduced as a fluorescent tracer into microfluidic devices containing living cells at day 7 of culture and time-sequential images were captured. Briefly, medium was aspirated from all reservoirs, 25 µl of sterile water was added into the reservoirs of one channel and 50 µl was added into the reservoirs of the other channel followed by 50 µl of fluorescent microbeads diluted 1:2,000 in sterile water. Sterile water was used instead of DPBS to avoid the formation of microbead aggregates. The generated hydrostatic pressure between the two medium channels resulted in luminal flow through the microvascular network. The time-sequential images were acquired every 10 s during 3 min using EVOS FL cell imaging system (Thermo Fisher, Nungambakkam, TamilNadu). Animated GIF images were then generated using Fiji software.

The fluorescent tracer was prepared by mixing 100 mg of powdered larval feed (O.range start-s, INVE aquaculture, Salt Lake City, UT, USA) with 150 µL of yellow-green 2.0 µL of FluoSpheres Carboxylate-Modified Microspheres (Thermo Fisher, F8827) and 50 µL of deionized water. The mixture was dried overnight at 25 °C in the dark and crushed into a fine powder.

For plunge freezing of HeLa-MuV cells under different stress conditions as described above, either a Vitrobot Mark 4 or Leica EM GP (Leica Microsystems) were used. Gold Quantifoil grids (R1/4, Au 200 mesh grid, SiO₂, Quantifoil Micro Tools) were glow-discharged and UV irradiated for 30 min for sterilization before being immersed in cell culture medium in 35-mm Ibidi μ-Dish. Next, HeLa-MuV cells were seeded in such dishes each containing 5-6 grids and cultured in an incubator overnight at 37 °C and 5% CO₂. Cells cultivated on grids were plunge-frozen in liquid ethane/propane mixture at close to liquid nitrogen temperature. The blotting conditions for the Vitrobot were set to 37 °C, 90% humidity, blot force 10, 10 sec blot time and 2 sec drain time and grids were blotted from the reverse side with the aid of a Teflon sheet from the front side. The blotting conditions for the Leica EM GP were set to 37 °C, 90% humidity, blot volume 3 μl, 2-3 sec blot time and grids were also blotted from the reverse side. For grids that were used in subsequent correlative imaging, 2 μl of 1-μm crimson beads (FluoSpheres carboxylate-modified microspheres, 625/645, Thermo Fisher Scientific) diluted 1:40 from original stock were added to the grid surface from one side before blotting. Grids were stored in liquid nitrogen until usage.

PECs were seeded in a 24 well plate (5 × 10⁵) and polarized for 72 h. In case of co-culture experiments, PECs were polarized for 24 h into M2-like macrophages and subsequently cultured for additional 24 h with OVA specific CD4⁺ Th1 cells. After washing three times with PBS, the cells were maintained in DMEM containing 10% FCS and either 1 mg/ml FITC-dextran (Sigma-Aldrich) or 1.25 × 10⁶/ml FluoSpheres™ Carboxylate-Modified Microspheres (2.0 μm; Thermo Fisher Scientific) to monitor pinocytosis and phagocytosis, respectively. Thereafter, PECs were harvested, stained with LIVE/DEAD® Fixable Yellow dye and analyzed by flow cytometry. Background values were determined upon co-incubation of cells with FITC labeled particles at 4°C and substracted from the values measured after co-culture at 37°C.