Exponentially growing cells were plated in 24-well microplates (BD Falcon) at a density of 2 ×105 cells per well in 400 µl of culture medium and were allowed to adhere overnight. Cells were then processed with or without N(G)-nitro-L-arginine methyl ester (L-NAME) as positive control, or growing concentrations of samples dissolved in the proper solvent, and incubated at 37 °C, 5 % CO2 for 24 hrs. The ultimate concentration of solvent in the culture medium was sustained at 0.5 % (v/v) to evade solvent toxicity. Then, cells were stimulated with 100 µg/ml lipopolysaccharide (LPS). After 24 hrs, cell-free supernatants were gathered and deposited at 80 °C until NO measurement using the Griess reaction (Green et al., 1990[9 (link)]) with slight changes. In the beginning, 100 µl aliquots of cell supernatants were incubated with 50 µl of 1 % sulphanilamide and 50 µl of 0.1 % N-1-naphtylethylenediamine dihydrochloride in 2.5 % H3PO4 at room temperature for 20 min. Then, absorbance at 540 nm was evaluated by an automated 96-well Varioskan Ascent plate reader (Thermo Electron) and the existence of nitrite was measured by comparison with an NaNO2 standard curve.
Varioskan ascent plate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Varioskan Ascent plate reader is a versatile instrument designed for a wide range of microplate-based assays. It is capable of performing absorbance, fluorescence, and luminescence measurements with high sensitivity and accuracy.
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4 protocols using varioskan ascent plate reader
1
Nitric Oxide Production Assay
2
Anti-inflammatory Activity Evaluation using RAW 264.7 Cell Line
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The anti-inflammatory activity was performed using the method reported by Ahmad et al. (2005[1 (link)]) with some modifications. RAW 264.7 cells (2×105 cells per well) were seeded at 400 µl of culture medium and incubated for 16 h at 37 °C and 5 % CO2 for adherence. Afterwards, cells were treated with extract dissolved in DMSO (0, 2.5, 5, 10, 20, 40 and 80 µg/ml) or the positive control (L-NAME) and then incubated for 24 h. As for the cytotoxicity assays, the DMSO final concentration in the culture medium was limited to 0.5 % (v/v). To release NO, cells were treated with lipopolysaccharide (LPS, 100 µg/ml) and incubated for 24 h at 37 °C under 5 % CO2. After that, 100 µl of cell supernatant were mixed with 100 µl of Griess reagent, prepared as described by Green et al. (1990[11 (link)]), and incubated at room temperature for 20 min. Nitrite quantification was estimated using NaNO2 standard curve and the absorbance was read at 540 nm using an automated 96-well Varioskan Ascent plate reader (Thermo Electron).
3
Nitric Oxide Production Assay in RAW 264.7 Cells
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Exponentially growing RAW 264.7 (murine macrophages) were plated in 96-well microplates (BD Falcon, Franklin Lakes, NJ, USA) at a density of 7.5 × 104 cells per well in 100 μL of culture medium (DMEM) and were allowed to adhere overnight. Cells were then treated with or without positive control N(w)-nitro-l -arginine methyl ester (l -NAME, ≥98%, Sigma–Aldrich), or increasing concentrations of extracts dissolved in DMSO, while the final concentration of solvent in the culture medium was maintained at 0.5% (v/v) to avoid solvent toxicity. Cells were then stimulated with 100 μg/mL lipopolysaccharide (LPS) and incubated at 37 °C, 5% CO2 for 24 h. After 24 h, cell-free supernatants were collected and immediately determined using the Griess reaction [52 (link)] with minor modifications. Briefly, 100 μL aliquots of cell supernatants were incubated with 50 μL of 1% sulphanilamide and 50 μL of 0.1% N-1-naphtylethylenediamine dihydrochloride in 2.5% H3PO4 at room temperature for 20 min. Absorbance at 540 nm was then measured using the automated Varioskan Ascent plate reader (Thermo Electron, Waltham, MA, USA) and the presence of nitrite was quantified by comparison with a NaNO2 standard curve.
4
Anti-inflammatory Activity of T. africana
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To investigate the anti-inflammatory activity of T. africana methanol extract, nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was examined. Exponentially growing cells were plated in 24-well microplates (BD Falcon) at a density of 2×10 5 cells per well in 400 µl of culture medium and were allowed to adhere overnight. Cells were then treated or not with positive control L-NAME or increasing concentrations of methanol extract dissolved in DMSO and incubated at 37 °C, 5% CO 2 for 24 h. The final concentration of solvent in the culture medium was maintained at 0.5% (v/v) to avoid solvent toxicity. Cells were then stimulated with 100 µg/mL LPS prepared from Escherichia coli. After 24 h, cell-free supernatants were collected and nitrite production was measured using the modified method of Green et al. [38] . Griess reagent (50 µL of 1% sulphanilamide and 50 µL of 0.1% N-1-naphthylethylenediamine dihydrochloride in 2.5% H 3 PO 4 ) was added in equal volume (100 µL) to cell supernatant and incubated at room temperature for 20 min. The absorbance at 540 nm was then measured using an automated 96-well Varioskan Ascent plate reader (Thermo Electron) and nitrite was quantified by comparison with a NaNO 2 standard curve. Triplicate measurements were carried out for all samples.
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