Luminex bio plex 200 system

Isolated neutrophils were incubated with 0, 50 or 200 nM PMA at 37°C for 45 minutes. After stimulation, the supernatant was collected and frozen down at -80°C until use. Samples were thawed and chemokine levels were measured in plasma using the Bio-Plex Pro Mouse Chemokine Assay (Bio-Rad), on a Bio Plex 200 Luminex System. Cytokine and chemokines with detectable supernatant levels were then analyzed by multiple T-testing, with correction for multiple comparisons, in order to determine the effects of PMA stimulation on neutrophil chemokine production.

Thawed plasma samples were utilized to determine N-Cadherin levels in all enrolled subjects. Samples were subject to centrifugation at 10000xg for 5 minutes at 4°C to eliminate any debris. N-Cadherin concentration was determined via a customized 11-plex multiplexing analysis kit (R&D systems, Minneapolis, MN, USA. Cat # LXSAHM) following manufacturer protocol. A standard protein mix, provided with the kit, was used to prepare a serial dilution. The results were obtained using the BioPlex 200-Luminex system (Bio-Rad, CA, USA). Analysis of the results were done using the BioPlex manager software. No significant cross reactivity with different proteins was observed. The confidence level between the expected and the observed standard concentration levels for N-Cadherin was between 95-105% as assessed by the system. The concentrations of the N-Cadherin in the study samples were calculated using a 5-Pt standard curve.

Determination of cytokine concentrations was carried out using multiplex cytokine assays conducted using the Bio-Plex 200 Luminex system (Bio-Rad, Hercules, CA, USA). At UOL, cytokines IL-1 $\beta$ , IL-10, IL-1ra, and IL-12 were measured, and at RIVM IL-12p70, IL-10 and IL-1 $\beta$ . Briefly, antibody-coated magnetic beads (50 µL) were loaded to a 96-well plate, and an equal volume of cell culture supernatant fractions were added, alongside multiplexed standard curves for relevant cytokines. Samples were incubated on a plate shaker for 2 h at room temperature. The plate was washed with wash buffer three times using an automated magnetic plate washer prior to the addition of biotinylated detection antibody (50 µL) and then incubated on a plate shaker for 1 h at room temperature. The plate was again washed three times prior to the addition of streptavidin-PE (50 µL) and incubation on a plate shaker for 30 min. The plate was then washed three times, and assay buffer (100 µL) was added to wells. The plate was analysed on a Bioplex 200 analyser using the recommended gating settings.

Blood plasma collected in lithium heparin tubes was used for blood chemistries. The IDEXX Catalyst One Analyzer (IDEXX, Westbrook, Maine, USA) clips included creatinine (CREA), creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate (LAC), and lactate dehydrogenase (LDH). Blood plasma specimens collected in EDTA tubes were evaluated for inflammatory markers. Initially, a 27-plex assay was evaluated to identify detectable analytes to down-select. Next, a customized rat four-plex cytokine/chemokine kit (Millipore, Burlington, MA, USA) on a Bio-plex^® 200 Luminex system (Bio-Rad, Hercules, CA, USA) was used for analyses according to the manufacturer’s instructions based on the analytes that had detectable levels from the 27-plex assay. The custom kit included interleukin (IL)-1β, IL-6, inflammatory protein-10 (IP-10), and leptin.