Conditioned media were collected after 7 days of culture and concentrated to 5× using Amicon Ultra centrifugal filter units (10 kDa, UFC501096, Merck). Concentrates were loaded onto gelatin zymogram gels (ZY00100BOX, Thermo Fischer) and processed according to the manufacturer's instructions. Developed gels were stained using SimplyBlue Coomassie stain (LC6060, Thermo Fischer).
Zy00100box
Manufactured by Thermo Fisher Scientific
The ZY00100BOX is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a standard-sized box containing the necessary components for the intended lab application. No further details on the core function or intended use of this product can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
3 protocols using zy00100box
1
Gelatin Zymography Protocol
2
Zymography of Osteoclast-Derived MMPs
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On day 9 of differentiation, media was changed to FBS-free media containing M-CSF and RANKL. Conditioned media was collected after 24 h, centrifuged, mixed with 2 volumes of Zymogram sample buffer (Bio-Rad) and loaded onto a 10% acrylamide gel containing co-polymerised gelatin (ZY00100BOX; ThermoFisher Scientific). Active recombinant human MMP8 and MMP9 (R&D systems) were used as positive controls. Gels were washed in renaturation buffer (BioRad) and then in development buffer (50 mM Tris, 10 mM CaCl2, 50 mM NaCl, 0.05% Brij 35, pH 7.6) to activate MMPs. Gels were processed for silver staining and scanned using an Epson V700 Photo scanner and Epsonscan software.
3
Zymography for Evaluating MMP Isoenzymes
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Zymography was used to assess the activity of different MMP isoenzymes. Proteins are separated in sodium dodecyl sulphate (SDS). After 9 days of osteoclast differentiation on acellular cartilage, the media was changed to FBS-free media with M-CSF and RANKL for 24 h. Conditioned media was collected, centrifuged to remove cell debris, and mixed with 2 volumes of Zymogram sample buffer (1610764; Bio-Rad). Samples were loaded onto a 10% acrylamide gel containing copolymerised gelatin (ZY00100BOX; ThermoFisher Scientific) run at 150 V for approximately 2 h. Active recombinant human MMP8 (908-MP-010, R&D systems) and MMP9 (911-MP-010, R&D systems) were used as positive controls. Gels were washed in renaturation buffer (1610765, BioRad) to remove SDS and then in development buffer (50 mM Tris, 10 mM CaCl2, 50 mM NaCl, 0.05 % Brij 35, pH 7.6) to remove renaturation buffer and activate MMPs. Gels were then processed for silver staining and scanned using an Epson V700 Photo scanner and Epsonscan software.
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