Hcytmag 60k px38

To test IFN and additional cytokine production, culture supernatants from mock and BKV infected RPTE and VEC at various time points were collected and stored at -80°C until processing. IFNβ levels were determined with the Human IFN-beta DuoSet ELISA kit (R&D Systems) following manufacturer’s instructions. A 38-plex human cytokine Luminex panel (HCYTMAG-60K-PX38, Millipore Sigma) was used to assay secretion of 38 additional cytokines. The collected supernatants were submitted to the Hillman Cancer Center (UHCC) Luminex Core Laboratory affiliated with the University of Pittsburgh Medical Center for the procedures.

500 uL live- and heat-killed bacteria-stimulated MoDC cell culture supernatant was harvested from 12-well tissue culture plates (Corning, Inc., Corning, NY, USA), centrifuged at 400× g for 10 min, recovered, aliquoted into pyrogen-free tubes (Fisherbrand, Waltham, MA, USA), and stored at –80 ℃. Adenosine triphosphate (ATP) levels were measured via colorimetric ATP assay kit (abcam, Cambridge, MA, USA), as per manufacturer instructions, and read on a Synergy Neo2 Hybrid Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA). Immune mediator levels in human MoDC cell culture supernatant were analyzed by multi-analyte bead-based immune assay (#HCYTMAG-60K-PX38, MilliporeSigma, Burlington, MA, USA), adapted for use with 96-well DropArray plates and DropArray LT210 washing station (both from Curiox Biosystems, Woburn, MA, USA), and read on a MAGPIX xMAP instrument (Luminex, Corp., Austin, TX, USA). N = 3–6 independent donors.
150 uL heat-killed bacteria-stimulated MoDC cell culture supernatant was harvested from 96-well U-bottom tissue culture plates after centrifugation at 400× g for 10 min at room temperature. Cell culture supernatant was recovered, aliquoted into a new, sterile 96-well U-bottom tissue culture plate, and stored at –80 ℃. Immune mediator levels in human MoDC cell culture supernatant were analyzed as outlined above. N = 6 independent donors.

BIMs were measured using the following ELISA kits: GFAP (Cat No: NS830, Sigma-Aldrich), NSE (Cat No: 420-85, IBL America), S100B (Cat No: EZHS100B-33K, MilliporeSigma), UCH-L1 (EH475RB, ThermoFisher), syndecan-1(RAB0736-1KT, MilliporeSigma) and MAP2 (EKU05950, BIOMATIK). The EIMs (including ICAM-1 and VCAM-1) were measured using a premixed multiplex assay (Cat. #HCVD2MAG-67K, MilliporeSigma). Systemic levels of CCs were measured using a 38-plex premixed immunological multiplex assay (HCYTMAG-60K-PX38, MilliporeSigma, Billerica, MA). CCs included CD40L, EGF, Eotaxin/CCL11, FGF-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GRO, IFN-α2, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, MCP-1, MCP-3, MDC (CCL22), MIP-1α, MIP-1β, TGF-α, TNF-α, TNF-β and VEGF. All experiments were performed according to manufacturer’s protocol. The levels of all markers are reported as pg/ml.