Protein concentrator

Bacteriophages were propagated on P. aeruginosa strains PAO1 or MRSN1680. The host bacteria were grown in heart infusion broth (HIB; Becton, Dickinson and Co., Franklin Lakes, NJ) supplemented with 5 mM calcium chloride and incubated in a vented culture flask at 37 °C and 200 rpm. Phage stock lysate was added to 250 mL of an early exponential phase bacterial culture grown in HIB (OD600 of 0.1–0.2; 10⁸ colony forming units/mL) at a multiplicity of infection of 0.1 (10⁷ plaque forming units/mL) and incubated in a 500-mL plastic Erlenmeyer flask at 37 °C and 200 rpm overnight. The phage and bacterial debris were pelleted by centrifugation at 5500× g overnight, and then the phage was purified with 1-octanol (Sigma, St. Louis, MO, USA) washes and a cesium chloride (Sigma) density gradient. The final phage concentrate was exchanged into gelatin-free SM buffer (100 mM sodium chloride, 50 mM Tris-HCl, pH 7.5, and 10 mM magnesium sulfate) by serial washes in a protein concentrator (Thermo Fisher, Waltham, MA, USA). Endotoxin levels were tested with the Endosafe-PTS device (Charles River Laboratories, Wilmington, MA, USA), and if needed, further purified using EndoTrap bulk resin (Hyglos GmbH, Bernried am Starnberger See, Germany) according to the manufacturer’s protocol, to ensure that the endotoxin level was below 500 EU per 10⁹ PFU (plaque-forming units).

LDH activity assays were performed as described previously [26 (link)]. Briefly, the supernatants from the cultures of ALS-As-iMNs and wild-type MNs were collected after a 4-week culture. Then, the supernatants were centrifuged, and the proteins in the supernatants were concentrated with protein concentrators (Thermo Scientific™). The LDH activity was measured in the concentrated supernatants with the LDH-cytotoxicity colorimetric assay kit II (BioVision). The genomic DNA (gDNA) in ALS-As-iMNs and wild-type MNs was extracted using the genomic DNA purification kit (Thermo Scientific™). The LDH activity values were normalized to the amount of gDNA of the ALS-As-iMNs and wild-type MNs.

HDMECs were washed once with phosphate-buffered saline (PBS) and placed in fresh PBS. The cells were irradiated with 20 J/cm² UVA (wavelength 320–400 nm, maximum peak 350 nm) using LZC-1 photoreactor system (Luzchem Research Inc. Ontario, Canada) or 50 mJ/cm² UVB (wavelength 290–320 nm, maximum peak 311 nm) using TL 20 W/12 RS UV lamps (Philips, Eindhoven, Netherlands). The time of irradiation were 119 min and 61 sec, respectively. Sham-irradiated HDMECs were rinsed and placed into the irradiator box for the appropriate time without UV irradiation. After irradiation, the cells were incubated in EGM for 24 h. The medium was collected and filtered through 0.2 μm filters (Millipore, Billerica, MA) to remove cellular components and debris. The medium was further concentrated by 60-fold with protein concentrators (pore size of 3 kDa, Thermo, Rockford, IL) and 50 µL of the concentrated CM in a total volume of 1.5 mL media was used for the treatment.