Bio plex 200 multiplex system

Ten tissue biopsies were chosen at random from acute and chronic ulcer patients and subjected to total protein extraction using the ProteoExtract Complete Mammalian Proteome Extraction Kit (MilliporeSigma, Burlington, MA, USA). The frozen tissue was ground using liquid nitrogen, transferred to a pre-chilled Eppendorf, and immediately subjected to total protein extraction. The supernatant of each sample was collected in a fresh microcentrifuge tube, and the concentration of total protein was quantified using Bradford’s protein assay reagent. The Bio-Plex Pro Human Cytokine multiplex assay (Bio-Rad) was used to quantify the levels of angiogenic markers in the tissue supernatants using a Bio-Plex 200 Multiplex System. A normalized concentration of protein was loaded to the wells and incubated with antibody as per the manufacturer’s experimental design. The concentration of each analyte measured using multiplexing was then normalized to the total protein concentration of that particular sample.

Ten stress related cytokines (IL-1β, IL-1RA, IL-6, IL-8, IL-10, IL-17, IP-10, MCP-1, MIP-1b, and TNF-alpha) were analyzed by using a commercial kit (Bio-Plex Pro Human Cytokine Group I 10-plex Assay, Catalog Number Y5002UN3DT, Bio-Rad) as per the manufacturer’s instructions. Fifty microliters of cleared saliva from each time point from all AC and YB participants were subjected to multiplex assay in duplicate. Cytokine levels were expressed as pg/mL based on the average fluorescent intensities as measured using Bio-Plex 200 Multiplex System (Bio-Rad) at the MUSC Proteogenomics facility.

All cytokine measurements were done with Bio-plex Pro technology on a Bio-plex 200 multiplex system (Biorad, Hercules, CA), detecting the cytokines interleukin (IL)-1β, IL-2, IL-10, IL-12p70, IL-13, IL-17A, GM-CSF, IFN-γ and TNF-α as per Biorad instructions. When a measurement failed to be above detection level defined by the standard curve, the lowest value in the data set served as a replacement instead of zero.

DuoSet enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN; BD Biosciences, Oxford, Oxon, United Kingdom) were used to assess serum cytokine levels according to the manufacturers’ instructions. Absorbance was read at 450 nm using a spectrophotometric ELISA plate reader (Anthos HTII; Anthos Labtec, Salzburg, Austria).
MILLIPLEX_MAP multianalyte panels (Merck Millipore, Watford, Herts, United Kingdom) were used for simultaneous detection and quantification of eight biomarkers and cytokines/chemokine in rat urine, including neutrophil gelatinase-associated lipocalin (NGAL), cystatin C, interleukin (IL)-18, monocyte chemotactic protein (MCP)-1, clusterin, calbindin, osteopontin, and kidney injury molecule (KIM)-1. The same technique was used to determine serum levels of IL-18. Assays were performed according to the manufacturer’s protocols. The plate was read on a Bio-Plex 200 multiplex system (Bio-Rad, Hemel Hempstead, Herts, United Kingdom). Urine tissue inhibitor of metalloproteinases-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) were analyzed by ELISA as described previously (6 (link)). Renal function (serum creatinine) was analyzed using the Jaffe assay by the Clinical Pathology laboratory at the Royal Free Hospital, London, United Kingdom. Where urine biomarkers were analyzed, matched creatinine values were used.

Cytokine protein levels in the BALF and serum were determined by multiplex immunoassay (Affymetrix, Santa Clara, CA). The assay was run on a Bio-Plex 200 multiplex system (Bio-Rad, Hercules, CA).