Primary osteoblasts (50,000 cells/cm2 in 12-well plate) were treated with 50 μg/ml of ascorbic acid for 8 h. Then, the cells were fixed with 4% PFA for 20 min at room temperature (RT), stained with 0.1% Sirius red (Sigma Aldrich) in saturated picric acid for 4 h and washed with PBS (Lonza). The stain was solubilized in 300 μL of destain solution (0.2 M NaOH/methanol 1:1) and the optical density was measured at 540 nm with a Synergy™ H4 instrument (BioTek Instruments, Inc.).
Synergy h4 instrument
Manufactured by Agilent Technologies
The Synergy H4 instrument is a multi-mode microplate reader designed for a variety of plate-based assays. It is capable of absorbance, fluorescence, and luminescence detection.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Sirius red, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Dc protein assay kit 2, by Bio-Rad (1 mentions)
Immobilon western kit, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Realtime glo, by Promega (1 mentions)
Vt1200 vibratome, by Leica (1 mentions)
Sta 396, by Cell Biolabs (1 mentions)
5 protocols using synergy h4 instrument
1
Quantitative Osteoblast Collagen Assay
2
Transwell Assay for Cell Migration and Invasion
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About 800 µL medium containing 20% FBS was added to the lower chamber of a transwell (8-µm pore size, Millipore, Billerica, MA, USA) and 5×104 cells in 200 µL of medium supplemented with 100 µM ZYX36-58 peptide or peptide solvent were seeded in the upper chamber with or without Matrigel (Matrigel: medium =1:6, 60 µL/well) for invasion and migration assays, respectively, and incubated as above. After culturing for 24 h (for migration assay of SKOV3 cells) or 48 h (for invasion assay of SKOV3 cells, and for migration and invasion assay of HO8910 cells), the chamber was fixed in 4% paraformaldehyde for 60 min, followed by staining with 0.1% crystal violet for 30 min, washing two times in PBS, and removing the cells in the surface of the upper chamber by cotton swab. Three randomly selected areas of the lower surface of the chamber were selected for photography under Axio Observer D1 fluroscence microscope (Carl Zeiss, Germany). Finally, the cells on the lower surface were lysed in RIPA buffer, and the absorbance at 562 nm of the cell lysate was analyzed by the BioTek Synergy H4 instrument.
3
Quantitative Western Blot Analysis of RANKL
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Total protein extraction, gel electrophoresis, transfer, and visualization were performed according to standard Western blotting procedures. Briefly, cell lysis was done in RIPA buffer and proteins were quantified using the DC Protein Assay Kit II (Biorad, Berkeley, CA) following the manufacturer's instructions, on a Synergy H4 instrument (BioTek Instruments, Inc., Winooski, VT). Twenty‐five micrograms of protein extracts were separated on a 10% Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS‐PAGE) and transferred to a nitrocellulose membrane. This was incubated with human RANKL monoclonal antibody (Alexis Corporation, Lausen, Switzerland), diluted 1:1,000 in 5% milk solution, for 2 hours at room temperature, then washed, probed with a secondary antibody conjugated with HRP and developed using the Immobilon Western kit (Millipore, Darmstadt, Germany). Images were acquired using the ChemiDoc MP Imaging System equipped with Image Lab Software (Biorad).
4
Tumor Slice Viability Assay
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Tumor slices were prepared as described previously (Sivakumar et al. 2019 (link); Nishida-Aoki et al. 2020 (link)). Briefly, dissected tumor tissues were molded into a 6-mm core using a biopsy punch. The cores were cut into 250-µm slices using Leica Vibratome VT1200. Slices were immediately placed on inserts in 24-well plates and incubated with Williams’ medium containing 12 mM nicotinamide, 150 nM ascorbic acid, 2.25 mg/mL sodium bicarbonate, 20 mM HEPES, 50 mg/mL additional glucose, 1 mM sodium pyruvate, 2 mM L-glutamine, 1% (v/v) ITS, 20 ng/mL EGF, 40 IU/mL penicillin, and 40 µg/mL streptomycin. After 24 h, slices were exposed to either DMSO (control), 200 nM staurosporine, 5 µM verteporfin, or 5 µM gemcitabine for 6 d. Overall tumor tissue viability was measured using real-time Glo (Promega) according to the manufacturer's instructions. Viability measurement was taken before (day 0) and after (day 6) drug treatment using a Synergy H4 instrument (Biotek).
5
Serum Biomarker Quantification Protocol
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For serum collection, blood was left at room temperature for 30 min. Blood clots were then removed by centrifugation at 1,000 g for 10 min in a refrigerated centrifuge. The supernatant was collected and IGFBP2 ELISA (Abcam ab207615), Triglyceride assay (Cell Biolabs STA-396) were carried out on samples according to the manufacture’s protocol. Absorbance was measured with Synergy H4 instrument (Biotek).
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