Quickplex sq 120 imager

Fecal pH and organic acids were assessed at V1, V2, and V3 using pH-indicator paper (Merck, Darmstadt, Germany) and validated LCMS, respectively (23 (link)). ELISA kits were used to analyze fecal biomarkers at V1, V2, and V3 including secretory immunoglobulin A (sIgA), calprotectin (Immundiagnostik AG, Bensheim, Germany) and alpha-1-antitrypsin (AAT) (BioVendor – Laboratorni medicina a.s., Brno, Czech Republic). Fecal cytokines were quantified as previously published (24 (link)), diluted 1:2 in Meso Scale Discovery diluent (MSD; Rockville, MD, United States), using V-Plex Plus kits and U-plex according to manufacturer’s instructions, and a QuickPlex SQ 120 Imager (MSD; Rockville, MD, United States). Total protein content in fecal extracts was quantified using Pierce BCA protein assay kit (Thermo Scientific) and used for cytokine level normalization.

The cocktail probes and their corresponding CYP-specific metabolites will be quantified in plasma using high-performance liquid chromatography/tandem mass spectrometry method, which was previously fully validated and described in [15] (link). The protein precipitation by acetonitrile will be used for plasma extraction.
Clinical chemistry analysis, including creatinine, AST, ALT, FBS, and HbA1c, will be analyzed, primarily at the Department of Medical Biochemistry at IMAM KHOMEINI hospital complexes.
The plasma levels of proinflammatory cytokines including IL-1β, IL-6, and TNF-α will be quantified by electrochemiluminescence immunoassays using the V-PLEX Proinflammatory Panel 1 Human Kit, QuickPlex SQ120 Imager, and WORKBENCH software (MSD, Rockville, MD). The genetic polymorphism of CYP450 enzymes including CYP2B6, CYP2C9, CYP2C19 and CYP2D6 will be evaluated. Genomic DNA will be extracted from whole blood samples throughout the study in both groups. DNA samples will be used for genetic analyses associated with mentioned CYP450s by using semi-quantitative RT-PCR or other appropriate methods [14 (link),15] (link).

Proinflammatory cytokines and chemokines in mouse lung and liver homogenates were determined using MSD mouse TH1/TH2 9-plex (Meso Scale Diagnostic #N05013B-1) or MSD prototype mouse 7-plex (Meso Scale Diagnostic #N75ZB-1) according to the manufacturer’s protocols and data were acquired in a MESO QuickPlex SQ 120 imager equipped with MSD Discovery Workbench 4.0.12 (LSR_4_0_12). Type I interferons (IFNs) in mouse lung and liver homogenates were quantitated using VeriKine High Sensitivity mouse IFN-α all subtype ELISA kit (PBL Assay Science #42115-1) or Verikine High Sensitivity mouse IFN-β ELISA kit (PBL Assay Science #42410-1). Optical density (OD) at 450 nm was measured using a Victor V multilabel reader (PerkinElmer) equipped with Wallac 1420 Workstation (Version 3 Revision 4).

Levels of 9 different immunoregulatory cytokines (IFN-γ, IL-1Β, IL-2, IL-4, IL-6, IL-8. IL-12P70, IL-13, and TNF-α) were assessed using a Mesoscale Discovery (MSD) U-plex 10 spot multiplex assay (Mesoscale discovery, Catalog # K15049K-1). After preparing the 96-well plate as per manufacturer instructions, normalized urine samples from 18 IC/BPS-HL, 18 age and sex matched IC/BPS-NHL counterparts, and 4 UC research participants were tested in duplicate. Plate readings were conducted using an MSD Quickplex SQ 120 imager (Model no: 1250) and the data used for analysis was obtained using the MSD workbench software program (version 3.0.18).
All patients involved in the study were screened prior to selection to ensure none of them were diagnosed with any inflammatory comorbidities that could act as confounding variables and potentially skew the results of the IHC or cytokine analyses. In addition, all patients were also screened to ensure they were not taking any medications that could impact biomarker expression at the time of sample acquisition. Lastly, medical professionals from the same Urology clinic who have been treating these patients for many years and are familiar with their IC/BPS history gave final approval for the enrollment and subsequent sample collection of all patients enrolled in this study.

Blood samples for the quantification of proinflammatory cytokines were kept on ice and rapidly sent to the research laboratory to be centrifuged at 1100× g (10 min at 4 °C) within 1 hour of sampling. Plasma was then aliquoted and stored at −80 °C until use. Levels of inflammatory markers INF-γ, IL-1β, IL-6, and TNF-α were quantified by electrochemiluminescence immunoassays using the V-PLEX Proinflammatory Panel 1 Human Kit, QuickPlex SQ120 Imager, and WORKBENCH software (MSD^®, Rockville, MD).

Circulating concentration of the cytokines TNFα, interferon gamma (IFN-γ) and interleukins (ILs) 4, 6 and 10 were quantified from blood samples utilizing a Meso Scale Discovery (MSD; Rockville, MD, USA) Rat Proinflammatory Panel 2 and a Quick Plex SQ 120 imager (MSD, Rockville, MD) using electrochemiluminescence technology.