Wound tissue was collected from each mouse and kept at -80˚C until processing. The wound samples were weighed and homogenized in RIPA buffer (Cell Signaling Technology, Inc.) and phosphatase inhibitor cocktails (MilliporeSigma). The samples were sonicated (20 kHz; Model VCX750; Sonics & Materials Inc., USA) three times (each time, 15 sec; interval time, of 10 sec), and centrifuged (Sorvall™ Legend™ X1R; Thermo Fisher Scientific, Inc.) at 13,416 x g for 10 min at 4˚C. The supernatants of each sample were used to determine the levels of total protein using the BCA protein assay kit (Thermo Fisher Scientific, Inc.), VEGF using the VEGF ELISA kit (mouse VEGF quantikine ELISA kit; cat. no. MMV00; R&D Systems, Inc.), and IL-6 using the IL-6 ELISA kit (mouse IL-6 quantikine ELISA kit; cat. no. M6000B; R&D Systems, Inc). Subsequently, the OD value of the solution in each well was measured using a colorimetric microplate reader (Model 860; Bio-Rad Laboratories, Inc.), setting the measured wavelength at 450 nm and the reference wavelength at 570 nm. The VEGF and IL-6 protein levels were expressed in units of pg/mg total protein. All standard solutions and samples were duplicated in a second plate (5 (link),16 (link)).
Model 860
Manufactured by Bio-Rad
The Model 860 is a compact and versatile centrifuge designed for a wide range of applications in laboratory settings. It features a durable and reliable construction, providing users with a consistent and efficient centrifugation process.
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Lab products found in correlation
Sorvall legend x1r, by Thermo Fisher Scientific (1 mentions)
Vegf elisa kit, by Thermo Fisher Scientific (1 mentions)
Il 6 elisa kit, by R&D Systems (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Mouse il 6 quantikine elisa kit, by R&D Systems (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Model vcx 750, by Sonics (1 mentions)
2 protocols using model 860
1
Quantification of Wound Tissue Proteins
2
SOD3 Enzymatic Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Levels of SOD3 were measured by ELISA according to manufacturers instructions (Uscn Life Science Inc. Wuhan, China). In brief, slices were homogenized in lysis buffer and protein concentration has been determined by the Bradford assay [18 (link)]. Lysates (100 µl) were added to the assay plate and incubated at 37 °C for 2 h. Thereafter, samples were consecutively incubated with solutions A, solution B, substrate and stop solution with washing steps in between (3 × 2 min each). Finally, the plate was read at 450 nm using a microplate reader (Biorad model 860) and enzyme activities were calculated against the standards provided with the assay and normalized to the protein concentration.
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