Cells were seeded at a density of an 8 × 103/well in 96-well cell culture plates 24 h before the treatment. After 24 h of treatment with 1, 2.5, 5, or 10 µM of HO-5114, the medium was discarded, and the cells were washed with phosphate buffered saline (PBS; Biowest, Nuaille, France) and fixed in 100 µL of a cold 10% trichloroacetic acid (TCA) solution (Sigma-Aldrich Co., Budapest, Hungary) for 30 min at 4 °C. After TCA was discarded, the cells were washed with a 1% acetic acid solution (Sigma-Aldrich Co., Budapest, Hungary) and dried overnight at room temperature. The next day, 70 µL 0.1% sulforhodamine B (SRB) (Sigma-Aldrich Co., Budapest, Hungary) in a 1% acetic acid solution was added to the wells for 20 min at room temperature. The plates were washed 5 times with a 1% acetic acid solution and dried for at least 2 h. Added to the cell was 200 µL of a 10 mM TRIS solution (Sigma-Aldrich Co., Budapest, Hungary) and the samples were incubated at room temperature on a plate shaker for 3 h. Absorbance was measured at 560 and 600 nm simultaneously using the GloMax®-Multi Instrument (Promega, Madison, WI, USA). OD600 was subtracted as the background from the OD560 values.
Trichloroacetic acid tca solution
Manufactured by Merck Group
Sourced in Hungary, Italy
10% trichloroacetic acid (TCA) solution is a lab equipment product. It is an aqueous solution containing 10% concentration of trichloroacetic acid. Trichloroacetic acid is a chemical compound commonly used in various laboratory applications.
Automatically generated - may contain errors
2 protocols using trichloroacetic acid tca solution
1
Sulforhodamine B Colorimetric Assay
2
Lipid Oxidation in Fresh and Cooked Meat
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The lipid oxidation of fresh and cooked LTL samples was evaluated in duplicate according to Siu and Draper (1978, slightly modified) . Minced sample, 2.5 g, was blended and homogenised with 12.5 mL of distilled water for 2 min at 9,500 rpm using an Ultra-Turrax tissue homogenizer (IKA, Germany). Before centrifugation, at 2,000 rpm at 4°C for 20 min, 12.5 mL of 10% trichloroacetic acid (TCA) solution (Sigma-Aldrich, Milan, Italy) was added.
The supernatant was collected after decantation through a paper filter (Whatman No. 541), and 4 mL of clear filtrate was transferred into 15-mL pyrex tubes; 1-mL 0.06 M 2-thiobarbituric acid (TBA, Sigma-Aldrich, Milan, Italy) was added and the samples were kept for 90 min in a water bath at 80°C; the samples were cooled before reading. At the same time, the blank was run (2-mL distilled water + 2-mL TCA solution + 1-mL TBA). Absorbance at 532 nm was measured against blank sample using a Jasco spectrophotometer (Model V550, UV/ VIS, Tokyo, Japan). Using 1,1,3,3 tetraethoxypropane (TEP, Sigma-Aldrich, Milan, Italy) as a standard, thiobarbituric acid reactive substances (TBARS) was expressed as milligram of malondialdehyde (MDA) per kilogram of muscle.
The supernatant was collected after decantation through a paper filter (Whatman No. 541), and 4 mL of clear filtrate was transferred into 15-mL pyrex tubes; 1-mL 0.06 M 2-thiobarbituric acid (TBA, Sigma-Aldrich, Milan, Italy) was added and the samples were kept for 90 min in a water bath at 80°C; the samples were cooled before reading. At the same time, the blank was run (2-mL distilled water + 2-mL TCA solution + 1-mL TBA). Absorbance at 532 nm was measured against blank sample using a Jasco spectrophotometer (Model V550, UV/ VIS, Tokyo, Japan). Using 1,1,3,3 tetraethoxypropane (TEP, Sigma-Aldrich, Milan, Italy) as a standard, thiobarbituric acid reactive substances (TBARS) was expressed as milligram of malondialdehyde (MDA) per kilogram of muscle.
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