Luminex 100

Sera were separated from clotted blood within 2 h of venesection and stored at −80°C. Serum concentrations of 48 cytokines (see Table S1 in the supplemental material) were assayed using Bio-Plex Pro 21- and 27-plex human cytokine, chemokine, and growth factor fluorescent bead-based assays (Bio-Rad Laboratories). Paired acute- and convalescent-phase samples were assayed together. In brief, antibody-coated fluorescent microspheres were incubated with manufacturer-supplied cytokine standards and study sera, washed, and incubated with detection antibody. Following washing, the microspheres were incubated with streptavidin-conjugated phycoerythrin before data acquisition on a Luminex-100 instrument (Bio-Rad Laboratories) using Bio-Plex Manager 4.1.1 software (Bio-Rad). Cytokine measurements below the detection limit of the assay were assigned values of the lower detection limit for each cytokine and were included in the analysis. At least one multiplexed assay failed in the case of five samples (3 acute, 2 convalescent), and these were excluded from analysis.

The cytokine levels in the serum were measured using the Human Cytokine Magnetic 25-Plex Panel kit (LHC009, Invitrogen) according to the manufacturer’s instruction and read on Luminex 100 (BioRad).

The skin samples were rinsed by cell wash buffer. A 500 mM phenylmethylsulfonyl fluoride in cell lysis buffer (0.5 ml) was prepared and added into test tubes with skin samples. The probe-type sonicator (GM70, Bandelin Electronic, Berlin, Germany) was used to homogenize the mixture for 10 s. Subsequently the mixture was freezed by liquid nitrogen. After thawing at room temperature, the mixture was homogenized by the sonicator for 10 s again. The sample was then centrifuged at 4500 xg for 4 min. The supernatant was collected and the cytokine concentration was quantified by using Bio-Plex Cytokine Assay Kit (Bio-Rad, Hercules, CA, USA) according to the method described previously [11 (link)]. The prepared sample was analyzed by Bio-Plex suspension array system (Luminex 100, Bio-Rad). The cytokines analyzed by Bio-Plex were interleukin (IL)-1β, IL-12, granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, and tumor necrosis factor (TNF)-α.

Concentrations of secreted cytokines and chemokines MCP1 (CCL2), KC (CXCL1), MIP-1α (CCL3), IL-6 and TNF-α in BAL were determined using a magnetic bead-based MILLIPLEX MAG multiplex assay (Millipore) and analyzed on a Luminex¹⁰⁰ (BIO-RAD, Munich, Germany). For this assay, BAL fluid was concentrated (10×) by ultrafiltration in Amicon Ultra-0.5 centrifugal filter devices (Millipore).

IL-6 was measured using ELISA mouse Ready-set-Go kit (Thermo Fisher Scientific; 88-7064-22, RRID:AB_2574986) as per the manufacturer’s instructions and read on a Safire II plate reader (Tecan). 2-Plex ProcartaPlex (Thermo Fisher Scientific; EPX02A-22187-901) was used to measure the concentrations of IFN-α/β, and the ProcartaPlex Cytokine and Chemokine Mouse 36-Plex (Thermo Fisher Scientific; EPXR360-26092-901) was used to assess the concentrations of 36 cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-12p70, IL-13, IL-18, TNFα, IL-9, IL-10, IL-17A [CTLA-8], IL-22, IL-23, IL-27, G-CSF [CSF-3], IFN-α, IL-3, IL-15/IL-15R, IL-28, IL-31, IL-1α, LIF, ENA-78 [CXCL5], M-CSF, Eotaxin [CCL11], GROα [CXCL1], IP-10 [CXCL10], MCP-1 [CCL2], MCP-3 [CCL7], Mip-1α [CCL3], Mip-1β [CCL4], Mip-2, and RANTES [CCL5]), read on a Luminex 100 (Bio-Rad; RRID:SCR_018025).