D1842

Cultured mouse ACCs were washed 3 times with cold standard extracellular bath solution, then incubated for 2 min with 50 µM dextran (10 and 40 kDa, Invitrogen, D1816, and D1842, respectively) in 37 °C 70 mM KCl containing external solution. Unbound dye was washed out with standard cold 0 Ca²⁺ immediately after incubation. The cells were then incubated in cold 0 Ca²⁺ solution. Z‐series of 1‐µm optical sections were scanned through the 40×oil‐immersion lens of a Zeiss 710 inverted confocal microscope. The consecutive optical sections were z‐projected and the total numbers of dextran fluorescent puncta per cell were determined. Images were processed with ImageJ (NIH).

10 kDa dextran conjugated to Alexa Fluor 647 (D-22914, Molecular Probes), 40 kDa dextran conjugated to Tetramethylrhodamine (D-1842, Molecular Probes) and 70 kDa dextran conjugated to Rhodamine B (D-1841, Molecular Probes), for the Dual Dye Assay – 70 kDa dextran conjugated to Oregon green (D7176, ThermoFisher Scientific) and 40 kDa dextran conjugated to Tetramethylrhodamine (D-1842, ThermoFisher Scientific), Alexa Fluor – 488 Phalloidin (1:200, A12379, ThermoFisher Scientific, A-12379 RRID:AB_2315147), VECTASHIELD with DAPI (H-1200, Vector Laboratories, RRID:AB_2336790).

Larval lymph glands were dissected in Schneider’s Drosophila Medium (Catalog number: 21720–024, Gibco). Lymph glands were then transferred to medium containing Oregon green conjugated 70 kDa dye (D7176, ThermoFisher Scientific) and Tetramethylrhodamine conjugated 40 kDa dye (D1842, ThermoFisher Scientific at a final concentration of 0.2 μg/μl). Lymph glands were imaged within 60 min of dye addition. Images were acquired from near the imaging surface to minimize out-of-plane fluorescence from dye in the medium. Comparable detection thresholds were ensured by setting the exposure level in the medium outside the primary lymph gland lobe to saturation level for both the dyes for image acquisition. PSC region was identified as the region just anterior to the first pair of pericardial cells which lie immediately posterior to the PSC cells.

The mice were cranially opened and injected with B16-mCerulean cells. After the metastases developed on day 14, the mice were intravenously injected with dextran [47 (link)] (0.1 mL of 10 mg/ml 40 kD TMR-dextran, D1842, Thermo Scientific, Waltham, MA, USA) and were then monitored with PE spinning-disc confocal system to detect the signal of melanoma cells, M/Ms, and vessels. The images were processed with ImageJ (Version 1.49, Fiji).

To characterize valve functionality, the assembly of the tissue chamber and the valve layers were bonded to a glass slide. A small PDMS slab (5 × 5 mm) was plasma-bonded on top of the valve inlet, and a hole was punched, through which blue food dye was injected. The valve maximum operating pressure was characterized by increasing the pressure in 6.9 kPa increments until the valve detached from the PDMS. Increasing membrane elevations were recorded with a USB digital microscope (Microcapture Pro 5MP, Celestron) from a side view of the valve. To study the elevation of the valve inside the tissue chamber, a finer characterization was conducted in steps of 0.7 kPa using a thin PDMS cylinder placed on the center of the valve. Micrographs were taken after pressure actuation.
To characterize the crosstalk between microchannels and detect any leaks between microchannels, dextran-conjugated rhodamine (40 kDa, D1842, Thermo Fisher) and fluorescein-5-isothiocyanate (389 Da, F7250, Sigma-Aldrich) were alternated in the 8 assay microchannels. For intercellular diffusion, fluorescent nuclear stains such as DAPI (62247, Thermo Fisher), Hoechst (H3570, Thermo Fisher) or Sytox Green (S7020, Thermo Fisher) were used in all assay microchannels. The valve was pressurized at 34.5 kPa.