To investigate the anti-inflammatory effect of the BM-hMSC secretome, we measured the amount of IL-10 secreted by BM-hMSC into the culture media by ELISA (Abcam, Cambridge, MA, USA) following the manufacturer’s instructions [17 (link)]. We explored the effect of IL-10 on steroidogenesis-related gene expression, androgen secretion, and pro-inflammatory marker expression in H295R cells after treatment with 0, 125, 250, or 500 pg/ml recombinant human IL-10 (rhIL-10; R & D Biosystem, Cat No. 217-IL-010). These concentrations were selected based on the previously reported level of IL-10 secreted by hMSCs [31 (link)]. H295R cells were then collected for gene expression analysis, and cell culture media were used for measurement of testosterone using an automated chemiluminescence immunoassay system, UniCel DxI 800, Access Immunoassay System (Beckman Coulter Inc., CA, USA) [30 (link)] and androstenedione using ELISA (Biovision, CA, USA) [32 (link)].
Elisa
Manufactured by Abcam
Sourced in United Kingdom, United States, Canada, China, France
ELISA (Enzyme-Linked Immunosorbent Assay) is a laboratory technique used to detect and quantify specific proteins or other biomolecules in a sample. It employs antibodies and color change to identify and measure the target analyte. The core function of ELISA is to provide a sensitive and reliable method for the detection and quantification of various analytes in a wide range of applications, such as diagnostics, research, and drug development.
Automatically generated - may contain errors
Lab products found in correlation
Elisa, by Thermo Fisher Scientific (7 mentions)
Elisa, by R&D Systems (7 mentions)
Elisa kit, by R&D Systems (4 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Elisa, by BioLegend (3 mentions)
Unicel dxi 800, by Beckman Coulter (2 mentions)
Access immunoassay system, by Beckman Coulter (2 mentions)
Inf γ, by R&D Systems (2 mentions)
Cxcl12, by R&D Systems (2 mentions)
Elisa kit, by Abcam (2 mentions)
Elisa, by BD (2 mentions)
Neutralizing antibody against mouse il 1α, by R&D Systems (2 mentions)
Duoset, by R&D Systems (2 mentions)
Elisa kit, by Cusabio (2 mentions)
Elisa, by Boster Bio (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Cytometric bead array, by BD (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
313 protocols using elisa
1
Anti-inflammatory effects of BM-hMSC secretome
2
Modulation of IFN-gamma and Tissue Factor in GM-CSF Macrophages
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siRNA duplexes (siRNAs) of extracellular signal-regulated kinase (ERK)-1, ERK-2, and CD206 were purchased from Santa Cruz Biotechnology. Transfection of siRNAs (50 nM) into GM-CSF-dependent macrophages on days 7 and 8 of culture was performed according to the manufacturer’s protocol for Lipofectamine™ RNAiMAX (Life Technologies). siRNA for GAPDH was utilized as a negative control.
On day 9, transfected cells (1 × 106) were stimulated with MPO (50 × 103 mU/L) for 6 h and then IFN-gamma protein levels in whole-cell lysates were determined by ELISA (Abcam Inc.) with an anti-IFN-gamma monoclonal antibody. The tissue factor protein level in whole-cell lysates was also determined by ELISA (Abcam Inc.) with an anti-tissue factor monoclonal antibody as an unrelated control.
On day 9, transfected cells (1 × 106) were stimulated with MPO (50 × 103 mU/L) for 6 h and then IFN-gamma protein levels in whole-cell lysates were determined by ELISA (Abcam Inc.) with an anti-IFN-gamma monoclonal antibody. The tissue factor protein level in whole-cell lysates was also determined by ELISA (Abcam Inc.) with an anti-tissue factor monoclonal antibody as an unrelated control.
3
Quantifying BMP-2 Release from Biomaterials
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To determine the cumulative release of growth factors at 1 h, 6 h, 12 h, 1 d, 3 d, 7 d, and 14 d, we placed the samples in vials containing 5 mL of α-minimum essential medium (α-MEM) (HyClone, Thermo, USA) at 37 °C to release growth factors. At each time point, 0.5 mL of medium was collected and the same volume of α-MEM was supplemented. The release of BMP-2 was detected by enzyme-linked immunosorbent assay (ELISA) (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Briefly, we coated the wells with standards and test samples, and blocked the remaining protein-binding sites in the coated wells with 5% non-fat dry milk. We added and incubated the following components in sequence: goat biotinylated detection polyclonal antibody against BMP-2, avidin-biotin-peroxidase complex, and TMB solution. After sufficient color development, we added stop solution to the wells and read the optical density at 450 nm. Finally, we prepared a standard curve and interpolated the concentration of test samples from this standard curve.
To determine the release of BMP-2 at 1 h, 6 h, 12 h, 1 d, 3 d, 7 d, and 14 d, at each time point, we placed the samples in vials containing 5 mL of α-MEM at 37 °C to release growth factors. At each time point, the culture solution was collected and 5 mL of fresh α-MEM was added. The release of BMP-2 was detected by ELISA (Abcam, Cambridge, UK).
To determine the release of BMP-2 at 1 h, 6 h, 12 h, 1 d, 3 d, 7 d, and 14 d, at each time point, we placed the samples in vials containing 5 mL of α-MEM at 37 °C to release growth factors. At each time point, the culture solution was collected and 5 mL of fresh α-MEM was added. The release of BMP-2 was detected by ELISA (Abcam, Cambridge, UK).
4
Quantifying Nitric Oxide Production in DCs
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The culture medium was obtained from LPS (0.1 μg/mL)- or vehicle-stimulated DCs (5 × 105 or 1 × 106 cells) in the absence or presence of Cl-amidine (50–200 μM). The levels of NO in the culture medium were measured by ELISA according to the manufacturer’s instructions (Intron Biotechnology, Seongnam, Korea). To assay NOS activity, recombinant NOS (180 ng iNOS; 210 ng eNOS; 420 ng nNOS; Enzo Life Sciences, New York, NY, USA) was incubated with or without Cl-amidine (200 μM) for 1 h, and then activity was measured for 1 h by ELISA, according to the manufacturer’s instructions (Biovision, Milpitas, CA, USA).
5
Measuring Leptin and Adiponectin Levels
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Leptin concentrations were determined by a multiplex assay (MMHMAG-44k, EMD Millipore, Billerica, MA, USA). The adiponectin concentration was determined by ELISA (BioVision, Inc.).
6
Quantification of IL-17, IL-22 and cAMP
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The culture supernatants of either CD4+ T cells or Th17 cells were harvested and analyzed for IL-17 and IL-22 concentrations with ELISA (eBioscience, USA) following the manufacturer’s instructions. The lysates of cultured Th17 cells were harvested and quantified for cAMP concentration with ELISA (Biovision, USA) according to the manufacturer’s instructions.
7
Serum Cortisol and Nitric Oxide Levels
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Serum cortisol was evaluated using ELISA (“Cortisol ELISA, ABCAM”), with an intra and inter assay coefficient of variation of 4.8 and 7.8 respectively and a sensitivity of 0.4 µg/dL. The reference range obtained by our laboratory from a control population was of
0.8-4.5 µg/dL (median: 2.1 µg/dL).
NO was measured as previously described by Cabrera Blatter et al. (2012), using ELISA (BioVision, EE.UU) and calculating the NO production by the nitrites and nitrates produced (NO nit/nit). The intra and inter assay coefficients of variation were 3.1% and 5.8% respectively with a sensitivity of 0.1 nmol nitrites/well. The reference range for our laboratory is 3.2 – 7.2 nmol/mL (median: 5 nmol /mL).
0.8-4.5 µg/dL (median: 2.1 µg/dL).
NO was measured as previously described by Cabrera Blatter et al. (2012), using ELISA (BioVision, EE.UU) and calculating the NO production by the nitrites and nitrates produced (NO nit/nit). The intra and inter assay coefficients of variation were 3.1% and 5.8% respectively with a sensitivity of 0.1 nmol nitrites/well. The reference range for our laboratory is 3.2 – 7.2 nmol/mL (median: 5 nmol /mL).
8
Metabolic Profiling of Murine Tissues
9
Quantification of Caspase-1 and IL-1β
10
Visfatin, Anti-CCP, and IgM-RF in Patients
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Fasting blood samples were collected from all patients at baseline and after three months. The samples were immediately centrifuged and stored at −20°C. The serum concentration of visfatin was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) (Biovision, Milpitas, California, USA) as described previously [14] (link). The levels of serum anti-cyclic citrullinated peptide antibodies (anti-CCP) and IgM rheumatoid factor (IgM-RF) were measured using a standard ELISA assay (Test Line S.R.O., Czech Republic). CRP and total and HDL-cholesterol were determined using routine laboratory techniques.
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