Syringe pump

Microfluidic cell culture was conducted in the three-layer microfluidic system described above. Prior to cell seeding, the microchannels were incubated with cell culture medium used for each cell type at 37 °C for at least 2 hours. For devices containing fibronectin-coated polyester membranes, 40 µg/mL fibronectin solution was introduced into the channels pretreated with corona point discharge and incubated at 37 °C and 5% CO2 for 30 minutes prior to incubation with cell culture medium. Next, cells suspended in culture medium at approximately 10 million cells/ml were injected into the upper channel (Fig. 1I) and allowed to settle and attach to the membrane surface under static conditions at 37 °C and 5% CO2 for 2 hours. Following microscopic examination to confirm cell attachment, the microchannels were gently flushed to remove non-adherent cells and then connected to syringe pumps (Braintree Scientific) that generated a flow of culture medium at volumetric flow rates of 70–100 µl/hour. Cells were cultured for 24–72 hours with continuous medium flow as needed to establish confluent monolayers (Fig. 1J) and for over 7 days in select experiments (Fig. S5). Cell viability was assessed by fluorescence microscopy imaging of cells labeled with calcein-AM and ethidium bromide homodimer according to standard protocols (Live/Dead kit, Invitrogen).

Gold(III) chloride trihydrate, sodium borohydride,
poly-di-(carboxylatophenoxy)phosphazene disodium salt (PCPP, 1 MDa), spermine,
reduced l-glutathione, hydrogen peroxide (H₂O₂),
ferrous perchlorate hydrate
(Fe(ClO₄)₂·xH₂O),
potassium dioxide (KO₂), and lipopolysaccharides (LPS) were purchased
from Sigma-Aldrich (St. Louis, MO). PEG-PLL (PEG M_W 5000/PLL
M_W 4900) was purchased from Alamanda Polymers. Recombinant human
interferon-γ protein (IFN-γ) was
purchased from Abcam. For the microfluidic setup, the herringbone mixer microfluidic
chip (channel diameter of 600 μm) and male luers were
purchased from Microfluidic ChipShop (Jena, Germany), the polyethylene tubing from
VWR (Philadelphia, PA), and the syringe pumps from Braintree Scientific (Braintree,
Ma). HepG2, RAW264.7, Renca, and SVEC4–10 cell lines were purchased from ATCC
(Manassas, VA). LIVE/DEAD assay kits were purchased from Invitrogen (Grand Island,
NY). Cells were cultured in Dubecco’s Modified Eagle Medium from Invitrogen
(Grand Island, NY) supplemented with 1% penicillin and 10% fetal bovine serum unless
specified otherwise. TNF-α ELISA kit was purchased from
Invitrogen (Grand Island, NY).
The experimental details for the synthesis of the arylboronate
polyphosphazene derivative (PPB) are detailed in the Supporting Information.

Mice were inoculated with 10⁸ CFU of UPEC strain CFT073 or K. pneumoniae strain KPPR1 in 50 μl of phosphate-buffered saline, as previously described^{18 (link)}. Mice were anesthetized with tribromoethanol (250 mg/kg). Inoculum was transurethrally-instilled in the urinary bladder with a polyethylene tubing (outer diameter 0.061 mm)-protected needle controlled by a syringe pump (flow rate=100 μl/minute, Braintree Scientific) to avoid vesicoureteral reflux. Urine was collected prior to euthanasia at 6, 24, 48 hours or 7 days post-inoculation by holding the animals above a sterile Petri dish. Organs were removed aseptically after euthanasia, weighed and homogenized. Urine and organ homogenates were diluted, plated, and incubated aerobically at 37°C to determine CFU/g or ml.