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The NR method is commonly used to determine the cytotoxicity of tested compounds. Neutral red (NR) is absorbed by living cells and bound in lysosomes. This dye is washed away from dead cells. In an acidified environment, the dye is released from the cells. The color intensity is directly proportional to the amount of dye captured and thus the number of viable cells [43 (link)]. The assay was performed on 96-well plates (100 µL 1 × 10⁵ cells/mL). After 24 hrs incubation with examined extracts or without them, the culture media was removed and 100 µL NR (40 µg/mL) was added. Incubation was performed for 3 hrs at 37 °C. After this time, the dye was removed and all wells were rinsed with 200 µL of 0.5% formalin in 1% CaCl₂. After 2 min, the solution was removed and 100 µL of 1% glacial acetic acid in 50% ethanol was added to all wells. Thereafter, we performed extraction for 20 min with shaking at room temperature. Then, a spectrophotometric measurement of absorbance was done at a wavelength λ = 550 nm using an E_max precision microplate leader (Molecular Devices) spectrophotometric reader.

The DPPH method allowed testing of the antioxidant properties of a given compound or preparation. It is based on determining the degree of reduction of the stable radical, DPPH. Antioxidants reduce DPPH molecules by attaching a hydrogen atom to a nitrogen atom with an unpaired electron. As a result of this reaction, hydrazine is formed, which is accompanied by a change in the maximum absorbance from λ = 515 nm to λ = 405 nm, thereby changing the color from purple to yellow [45 (link)]. The assay was carried out in plastic 96-well plates. Dilutions of tested substances and Trolox in methanol were prepared. Trolox was the positive control and the standard. The negative control was methanol. Then, 100 mL dilutions of test substances, Trolox, and methanol were added to the wells. Then 100 μL of a previously prepared solution of DPPH (solution of DPPH in methanol at a concentration of 0.2 mg/mL) was added to all wells. After 10 min of incubation at room temperature, the absorbance was determined spectrophotometrically at λ = 515 nm using an E_max precision microplate leader Molecular Devices spectrophotometric reader.