The viability of SMCs was determined by MTT assay (Beyotime). Briefly, SMCs were seeded into 96‐well plates at a density of 5000 cells/well. Following exposure to stimulation, SMCs were incubated with 10 µL MTT (5 mg/mL)/well reagent for 4 hours at 37°C. The supernatant was carefully removed, and 75 µL/well dimethyl sulphoxide (DMSO) was added to dissolve the formazan crystals. Samples were then analysed at 570 nm using a Varioskan Flash multifunction plate reader (Thermo Scientific).
Varioskan flash multifunction plate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Varioskan Flash is a multifunction plate reader designed for a variety of spectroscopic measurements. It is capable of absorbance, fluorescence, and luminescence detection in microplates.
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Lab products found in correlation
10 protocols using varioskan flash multifunction plate reader
1
Viability Assessment of Smooth Muscle Cells
2
Quantification of Cytokine Secretion
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Cell culture supernatants were obtained by centrifugation at 1,000 × g for 10 min at 4°C. Secretion of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)) was determined using ELISA kits according to the manufacturer's protocols (catalog no. #070133, Uscn Life Sciences, Inc., Wuhan, China and catalog no. FHK0016, Jiamay Biotech Co. Ltd., Beijing, China, respectively). All operations were performed at room temperature. Mean absorbance for standards and samples was assessed in duplicate. The color reaction was detected using a Varioskan Flash multifunction plate reader (Thermo Fisher Scientific, Inc.).
3
Lung Injury Assessment Protocol
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After 4 h of ventilation treatment, the lungs were lavaged with 0.3 ml PBS by tracheal intubation and pumped back after 3 s. The same operation was repeated three times in a total volume of 0.9 ml PBS, and the recovery rate was 90%. The lungs were blotted dry with filter paper, weighed to obtain the wet weight, and then were dried in an oven at 65°C for 48 h to obtain the dry weight. The lung wet/dry (W/D) ratio was calculated. Protein concentrations were determined using the BCA protein assay (ComWin Biotech, China). myeloperoxidase (MPO) activity, an indicator of the neutrophils and macrophages infiltration, was determined using test kits obtained from Nanjing Jiancheng Bioengineering Institute, China. The enzymatic activity was assessed by measuring the changes in the absorbance at 450 nm using a Varioskan Flash multifunction plate reader (Thermo Scientific, IL, United States).
4
Lung Tissue Protein Extraction and OT Quantification
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The total protein of lung tissue was extracted and concentrations were assessed in the same manner as described as Western blot. OT was measured using ELISA kits following the manufacturer’s protocol. The optical density of the microplate was read at 450 nm using a Varioskan Flash multifunction plate reader (Thermo Scientific).
5
Quantifying Vitreous Biomarkers
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NLRP3, VASH2, VEGF-B, and CTGF in the vitreous samples were evaluated using Sandwich enzyme-linked immunosorbent assay (ELISA) (MyBioSource ELISA kit, San Diego, CA, and RayBiotech ELISA kit, Norcross, GA) according to each manufacturer's protocol. After color development, the optical density was assayed at 450 nm using a Varioskan flash multifunction plate reader (Thermo Fisher Scientific, Waltham, MA, USA). This experiment was independently repeated three times.
6
Quantification of Inflammatory Cytokines
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The levels of TNF-α, IL-1β and IL-6 in the cultured cell medium and mouse plasma were determined by use of the ELISA kit (Uscn Life Science Inc., Wuhan, China). Mean absorbance was assessed in duplicate and all operations were performed at room temperature. The color reaction was detected by use of Varioskan Flash multifunction plate reader (Thermo Scientific, Rockford, USA).
7
Inflammatory Cytokine Quantification in LDH Patients
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We obtained the serum sample from the patients with LDH and stored at −20°C. The supernatants harvested from NP cell culture and stored at −20°C until assay. The serum concentration of TNF-α was measured by ELISA according to the manufacturer’s instructions (Abcam, USA), IL-6 in the supernatants from conditioned NP cells cultures were detected utilizing Human IL-6 Elisa kit (BlueGene, China) follow the instructions protocol. The color reaction was assayed at 450 nm using a Varioskan flash multifunction plate reader (Thermo Fisher Scientific, Waltham, MA).
8
Quantifying IL-17A in T2DM Samples
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IL-17A concentrations in PBMCs and vitreous fluid from patients with T2DM and healthy controls were determined using the Human IL-17A Quantikine ELISA kit (BMS2017; eBioscience, San Diego, CA, USA). All procedures were conducted at room temperature, and the mean absorbances of the standards and samples were determined from duplicates. The reaction was assayed at 450 nm using a Varioskan flash multifunction plate reader (Thermo Fisher Scientific, Waltham, MA, USA).
9
Quantification of Inflammatory Mediators
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The cell culture supernatant was obtained by centrifugation at 1000 g for 10 min. The HMGB1 release, TNF-α and IL-1β secretion were determined by use of the ELISA kit (Uscn Life Science Inc., Wuhan, China) according to the manufacturer’s instructions. All operations were performed at room temperature. Mean absorbance for standards and samples was assessed in duplicate. The color reaction was detected by use of Varioskan Flash multifunction plate reader (Thermo Scientific, Rockford, USA).
10
Th17 and IL-17 in Osteonecrosis of the Femoral Head
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The circulating levels of Th17 and IL-17 were evaluated in the blood samples. Peripheral blood was obtained from 34 patients (six for stage 1, 13 for stage 2, six for stage 3, and nine for stage 4) with ONFH and nine controls with transcervical femoral fracture. The samples were analysed using a Human Th17 ELISA kit (eBioscience, BMS2017HS) following the manufacturer's instructions. All procedures were performed at room temperature, and the mean absorbance of the standards and samples was detected in duplicate. Colorimetric reactions were analysed at 450 nm on a Varioskan flash multifunction plate reader (Thermo Scientific, Germany). All the experiments were repeated at least three times.
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