The effects of CSEO, methyl isothiocyanate, hexadecanoic acid, and limonene on the induction of apoptosis were evaluated by the colorimetric protease (Sigma, Germany) method according to the manufacturer recommendations. In this way, the caspase-3-like activity level was measured based on the rate of color spectrophotometric produced through the release of a molecule (pNA attached to the substrate) under the enzyme caspase-3 activity. In brief, the promastigotes (1 × 106) were incubated with CSEO, methyl isothiocyanate, hexadecanoic acid, and limonene at the concentrations of 6.25, 12.5, and 25 µg/mL for 24 h and were centrifuged at 700 rpm for 5 minutes at 4°C. Next, the cell residue was lysed, and the cell lysate was centrifuged again at 20,000 rpm for 10 minutes. Lastly, the supernatant of reaction (5 μl) was added to the 85 μl of buffer and 10 μl of caspase-3 (pNA-DEVD-Ac) solution and the mixture was incubated for 120 min at 37°C. The caspase-3-like activity was determined through the light absorption at 405 nm with the ELISA reader [27 (link)].
Colorimetric protease
Manufactured by Merck Group
Sourced in Germany
Colorimetric protease is a laboratory equipment used to measure the activity of proteases, which are enzymes that break down proteins. It provides a quantitative assessment of protease activity through colorimetric detection methods. The core function of this product is to enable accurate and reliable measurement of protease levels in various samples, supporting research and analysis applications.
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Lab products found in correlation
4 protocols using colorimetric protease
1
Apoptosis Induction by CSEO, Compounds
2
Caspase-3-like Activity in L. major Promastigotes
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Caspase-3-like activity of L. major promastigotes treated with RJ, 10-H2DA, 10-HDAA, and sebacic acid was assessed through the colorimetric protease (Sigma, Germany) technique according to the manufacturer guidelines. In this way, the caspase-3-like activity level was measured based on the rate of color spectrophotometric produced through the release of a molecule (pNA attached to the substrate) under the enzyme caspase-3 activity. Briefly, the promastigotes (106) were incubated with RJ, 10-H2DA, 10-HDAA, and sebacic acid for 48 h and were centrifuged at 700 rpm for 5 minutes at 4°C. In the next step, the cell residue was lysed, and the cell lysate was centrifuged again at 20,000 rpm for 10 minutes. Lastly, supernatant of reaction (5 μL) was added to the 85 μL of buffer, and 10 μL of caspase 3 (pNA-DEVD-Ac) solution and the mixture was incubated for 120 min at 37°C. The caspase-3-like activity was determined through the light absorption at 405 nm with the ELISA reader [29 (link)].
3
Caspase-3 Activity in Protoscoleces Treated with ECEO
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The activity of the Caspase-3 enzyme of protoscoleces treated with ECEO was determined according to the colorimetric protease (Sigma, Germany) protocols. This method was performed according to the spectrophotometric color formed by the discharge of a molecule (pNA attached to the substrate) by the enzyme caspase-3 activity. Briefly, the protoscoleces were exposed with ECEO (2.5, 5, and 10 µl/ml) and its components, 1,8-Cineole alone (2.5 and 5 µg/ml) days and were centrifuged at 650 rpm for 6 min at 4 °C, the cell residue was lysed, and centrifuged at 20,000 rpm for 10 min. Then, 5 μg of superior phase was mixed with buffer (85 μl) and caspase 3 (10 μl) (pNA-DEVD-Ac) solution and was kept for 2 h at 37 °C. Lastly, the absorption was assessed by the ELISA reader at 405 nm (Ezzatkhah et al., 2021 ).
4
Caspase-3-like Activity Assay in Promastigotes
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In order to determine the Caspase-3-like activity of promastigotes treated with ZSCME, the colorimetric protease (Sigma-Aldrich, Darmstadt, Germany) method was applied based on the manufacturer recommendations. The method was conducted according to the rate of color spectrophotometric formed through the release of a molecule (pNA attached to the substrate) under the enzyme caspase-3 activity. In summary, after incubating the promastigotes 106 with ZSCME for 48h, they were centrifuged at 650 rpm for 5 min at 4 °C, the cell residue was lysed, and the cell lysate was centrifuged again at 20,000 rpm for 10 min. Finally, supernatant of reaction (5 μL) was added to the 85 μL of buffer and 10 μL of caspase 3 (pNA-DEVD-Ac) solution and the mixture was incubated for 120 min at 37 °C. The caspase-3-like activity was measured by means of the light absorption at 405 nm with the ELISA reader [26 (link)].
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