ELISA was used to analyse the interaction of SspA-1, SspA-2 with C3a, C5a protein as described previously [20 (link)]. For C3a and C5a bound assay, 0–7 μg/mL SspA-1T, SspA-2T and BSA as a negative control were immobilized to 96-well plate (Corning, USA), 5 μg/mL C3a and C5a were applied after blocking with 1% BSA in PBST (PBS with 20% Tween-20), respectively. Mouse C3a/C3a des Arg monoclonal antibody (Abcam, England) or rabbit C5a monoclonal antibody (Abcam) was applied at 37 °C for 1 hour to test the direct binding capacity. For competitive binding assay, 5 μg/mL SspA-1T, SspA-2T and BSA were immobilized to 96-well plate, 5 μg/mL C5a combined with 0–20 μg/mL C3a were applied after blocking with 1% BSA in PBST, respectively. Mouse anti-C5 monoclonal antibody (Proteintech, Wuhan, China) was applied at 37 °C for 1 hour to test the C5a binding capacity. The HRP-conjugate goat-anti-rabbit IgG (CST) or HRP-conjugate rabbit-anti-mouse IgG (CST) was used as the second antibody at 37 °C for 1 h before Chromogenic reaction by TMB Chromogenic Reagent kit (Sangon) and detected by SynergyTM H1 Hybrid Reader (Biotek, USA). These data were presented as means ± standard deviations (SD) from three replicates. The experiments were repeated for three times.
Synergytm h1 hybrid reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy H1 Hybrid Reader is a multimode microplate reader designed for versatile detection of various assays. It combines absorbance, fluorescence, and luminescence detection modes in a single instrument.
Automatically generated - may contain errors
4 protocols using synergytm h1 hybrid reader
1
ELISA Analysis of SspA-1, SspA-2 Interaction with C3a, C5a
2
Quantifying COX-Mediated PGE2 Synthesis
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Inhibition of COX activity was determined by measuring the synthesis of PGE2 according to the instructions provided with the kit. Brief, COX-1 or COX-2 enzyme and heme were added to test tubes containing COX reaction buffer (total of 0.5 mL). The mixture was vortex mixed and exposed to vehicle (DPBS) or CAPE (10 mg/mL), quercetin or myricetin (10 μM) for 10 min at 37°C. This was followed by the addition of AA (100 μM) with further incubation for 2 min. Hydrochloric acid (1 M) was added to stop the COX reaction followed by chemical reduction with stannous chloride solution. A standard curve with PGE2 was generated at the same time and from the same plate and was used to quantify PGE2 levels produced in the presence of test compounds. Absorbance was read using a plate reader (SynergyTM H1 Hybrid Reader, Biotek, United States) at 412 nm. Diclofenac (10 μM) was used as the reference compound, and all compounds were diluted in a buffer kit.
3
Evaluating Platelet Viability with MTT Assay
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The MTT assay was performed according to Mosmann (1983) (link). Using 96-well plates, platelets (1.5 × 108 platelets/mL) were incubated with CAPE (0.25, 0.5, 1, 2.5, 5, and 10 mg/mL) or quercetin or myricetin (10 μM) for 5, 15, 30, and 60 min. After the incubation period, platelets were incubated with 5 mg/mL of MTT solution for 3 h in a CO2 incubator. Then MTT dye was removed and 100 μL of solubilization solution (SDS 10% acidified) were added to the wells. Absorbance was measured at 540 nm using a microplate reader (SynergyTM H1 Hybrid Reader, BioTek, United States). The positive control was 10% triton-X.
4
Quantification of Methylglyoxal and AGEs in Serum
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Peripheral blood (0.5 ml) was collected by the intracardiac puncture. Blood was centrifuged at 5,000 × g for 10 min at 4°C and serum was transferred to microcentrifuge tubes. To measure MGO levels in serum, the samples were deproteinized using Deproteinizing Sample Preparation Kit - TCA (Abcam ab204708) according to the manufacturer’s instructions. Serum levels of MGO were measured using ELISA competitive kit for OxiSelect™ Methylglyoxal (Catalog No. STA-811, Cell Biolabs, San Diego, CA, United States). F-AGEs in serum were measured diluting the samples 1:2 in phosphate-buffered saline (PBS, pH 7.4), and transferred to black 96-well plates, 200 µl total per well. Fluorescence spectra were recorded in duplicate on a fluorometer (SynergyTM H1 Hybrid Reader, Biotek, United States). The excitation and emission wavelengths were 360 and 447 nm, respectively, and the PBS solution was used as blank. Individual concentrations were normalized by blank fluorescence. The fluorescence intensity was expressed in arbitrary units (a.u.). To assess glucose concentration, animals fasted for 8 h, blood from the tail vein was collected and glucose was measured using ACCUCHECK Blood Glucose (Monitoring System®, Roche Diagnostics, Indianapolis, United States).
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