After an overnight fasting for at least 10 hours when DK/DKA resolved, fasting blood samples were collected and centrifuged, and sera were collected for cytokine determination. The following cytokines were measured: interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17A/CTLA-8, IL-17F, IL-21, IL-22, IL-23, interferon-γ (IFN-γ), programmed death-1 (PD-1), PD ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4/CD152), CD40, transforming growth factor β1 (TGFβ1), TGFβ2, and TGFβ3. All these measurements of cytokines were performed using Milliplex MAP technology in the Luminex platform (Millipore, Billerica, MA). Immunology multiplex assay kits (HTH17MAG-14K-12, TGFBMAG-64K-03, and HCKPMAG-11K-04) were obtained from MERCK (MERCK Millipore Co., Billerica, MA, USA). All the collected serum samples were mixed and diluted at a ratio of 1:2 with buffer, and all samples, standards, and quality controls were assayed in accordance with the manufacturer’s instructions. Median fluorescent intensity (MFI) data were acquired using MILLIPLEX Analyst.V5.1 software, and a 5-parameter logistic method was used to calculate cytokine concentrations.
Milliplex map technology
Manufactured by Merck Group
Sourced in United States
Milliplex MAP technology is a multiplex assay platform developed by Merck Group. The core function of this technology is to enable the simultaneous detection and quantification of multiple analytes from a single small sample. The Milliplex MAP system utilizes color-coded magnetic beads coated with specific capture antibodies to perform multiplexed immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Luminex platform, by Merck Group (1 mentions)
Tgfbmag 64k 03, by Merck Group (1 mentions)
Ab6584, by Abcam (1 mentions)
Microtainer serum separator tube, by BD (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Primepcr technology, by Bio-Rad (1 mentions)
Cfx connect thermocycler, by Bio-Rad (1 mentions)
Luminex system, by Merck Group (1 mentions)
Iscript, by Bio-Rad (1 mentions)
4 protocols using milliplex map technology
1
Cytokine Profiling in Diabetes
2
Histochemical Analysis of Ocular Tissues
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Eyes were fixed for 48 hours in 4% (w/v) paraformaldehyde solution, rinsed in PBS, and stored overnight in 3% (w/v) sucrose in PBS. Deparaffinized sections of 7 μm thickness were stained with either hematoxylin and eosin or antibodies specific for pan-laminin (NB600-680, Novus), fibronectin (ab6584, Abcam), collagen IV (1340-08, Sanbio), or Iba-1 (019-19741, Wako). Vitreous detection of CCL-2 was performed using a custom Mesoscale Discovery ELISA assay. Detection of the remaining cytokines was performed using MILLIPLEX® MAP technology (Millipore). Four untreated eyes as well as two lipopolysaccharides treated eyes (100 ng/eye) were included as controls [25 (link)]. Statistical differences were evaluated using the Mann-Whitney U test.
3
Investigating Inflammatory Biomarkers in Patients
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Venous blood samples from all patients and healthy controls were collected for investigating hemoglobin levels, serum CRPs, and IL-6 levels. Sera were stored in aliquots at −80°C until tested. Twenty-four hour urine was collected for urinalysis. Serum hs-CRP levels, complement component (C3, C4) levels were measured using a Nephelometer (BN Prospec, Dade Behring, Germany). Serum cytokine levels were detected by bead-based MILLIPLEX MAP technology (Millipore Corporation, Billerica, MA, USA.) in all patients and healthy controls.
4
Quantifying Chemokine and C3 Expression
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Tissues were harvested and homogenized in phosphate-buffered saline (PBS) containing 1x Calbiochem protease inhibitor cocktail (Millipore Sigma). Serum was obtained by collecting blood from the facial vein into BD microtainer serum separator tubes. Tissue homogenates and serum were clarified by centrifugation at 10,000×g. Chemokine concentrations were determined using a BioRad Luminex system and Millipore Milliplex MAP technology. Concentrations of C3 were measured by ELISA (Abcam, Cat. # ab157711).
Gene expression studies were performed using RNA isolated from tissue and cells using the Trizol (ThermoFisher) method and converted to cDNA with iScript (BioRad). Real-time PCR was performed using PrimePCR technology with commercially validated primer sequences (Biorad) and a Biorad CFX-Connect thermocycler as directed. Relative expression was calculated using the 2–ΔΔCt method with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as a reference gene. Final PCR products were resolved on a 2% agarose gel and imaged with an Azure Biosystems C-series gel documentation system (Dublin, CA) to confirm C3 expression in cell subsets.
Gene expression studies were performed using RNA isolated from tissue and cells using the Trizol (ThermoFisher) method and converted to cDNA with iScript (BioRad). Real-time PCR was performed using PrimePCR technology with commercially validated primer sequences (Biorad) and a Biorad CFX-Connect thermocycler as directed. Relative expression was calculated using the 2–ΔΔCt method with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as a reference gene. Final PCR products were resolved on a 2% agarose gel and imaged with an Azure Biosystems C-series gel documentation system (Dublin, CA) to confirm C3 expression in cell subsets.
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