Ptfe tubing

To fabricate the reservoir, two needles of 200 µm in diameter were inserted into a 0.5 mm thick piece of round PDMS mold with the diameter of a single well in a 48-well plate. The needles were placed such that they run perpendicular to the flat surface of the PDMS mold, and the PDMS/needles were placed into a single well of a 24-well plate. Unpolymerized PDMS was poured to fill the well, covered the PDMS mold, and left to cure at 60°C overnight.
The cured 24-well PDMS was removed using the fine-tipped weighing spoon as described earlier. The needles were removed, and the 0.5 mm thick 48-well PDMS mold was removed using the weighing spoon. The top half of the 24-well PDMS block was removed using a sharp blade. In total, two pieces of 150 mm long PTFE tubing (Cole-Palmer, Vernon Hills, IL, United States) were inserted into each of the channels created by the needles in the 24-well PDMS cylindrical block, and one of the tubings was threaded through one of the two channels such that a 30 mm length of tubing extends from the bottom end of the 24-well block. The other tubing was threaded through the second channel such that the end does not extend past the bottom opening (Supplementary Figure S1). The 24-well PDMS block with tubing was plugged into a capless 5 ml microfuge tube (Eppendorf) and sealed using PDMS. The reservoir was then heat sterilized at 121°C and 15 psi.

The BMDM circuit was
printed using a modified, custom-made printer (iotaSciences Ltd, Oxford,
U.K.). The three-axis traverse on the printer holds a 25 G dispensing
needle (Adhesive Dispensing Ltd) connected by PTFE tubing (Cole-Parmer)
to a 250 μL glass syringe (Hamilton) controlled by a syringe
pump (iotaSciences Ltd). The dispensing needle was brought 300 μm
above the surface of the dish and then infused (10 μL/min) DMEM
+ 10% FBS + 10% L929 + 1% P/S + 2% fibronectin as it drew the circuit
[3]. Once completed, the circuit was overlaid with 2 mL of FC40 (iotaSciences
Ltd) to prevent evaporation. Circuits were printed on both 60 mm tissue
culture-treated dishes (Corning, 430166) and 60 mm suspension dishes
(Corning, 430589). As BMDMs are extremely adherent, suspension dishes
were used when cells were to be extracted (Figure S3).

The microfluidic device was comprised of a molded poly(dimethylsiloxane) (PDMS, General Electric RTV 650 Part A/B) slab bonded to a glass substrate. High-resolution printing (5080 dpi) was used to print the mask with the design pattern on a transparency film. The mask was used to fabricate 50 µm high SU-8 2050 photoresist (Microchem) features on a silicon wafer via photolithography. PDMS molds with embossed channels were fabricated using soft lithography by curing the pre-polymer on the silicon master for 2 hours at 70°C. The PDMS replica was then peeled off the silicon master. Inlets and outlets for the fluids and cells were created in PDMS using a steel punch. The surface of the PDMS replica and a clean glass coverslip (Fisher Scientific) were treated with air plasma for 90 seconds (Model PDC-001, Harrick Scientific) and irreversibly bonded to complete the device assembly (Figure S7). The device inlets were then connected to 1 mL syringes (BD Biosciences) with 23 G ¾ size needles (BD Biosciences) via PTFE tubing (Cole-Parmer). All syringes were calibrated and pushed by a constant pressure syringe pump (Harvard Apparatus). Prior to each experiment, the device was also loaded with fibronectin (25 µg/mL, Invitrogen) and kept at room temperature for 30 minutes to promote optimal cell adhesion.

Plasma was separated from whole blood by the sedimentation of the red blood cells (RBCs) in the trenches of the microfluidic devices. Before using, the devices were washed three times with IPA and twice with PBS. A syringe pump (Chemyx, Fusion Touch, version 1.7.6 c) was used to control the flow, using either positive or negative pressure, in the 1–15 µL min⁻¹ range at a constant rate. In addition, 250 µL syringes (Hamilton, Reno, NV, USA) were used to introduce the sample into the devices. PTFE tubing (0.22 in ID × 0.042 in OD, Cole-Parmer, Vernon Hills, IL, USA) and capillary tubing (polymicro flexible fused silica capillary tubing, ID 50 µm, OD 375 µm, Lisle, IL, USA) were fixed with UV-curable resin (DecorRom, Shenzhen, China) to connect the microfluidic devices to the syringe pump. The entire process is summarized in Figure 1.