Sodium Phosphate Buffer (25mM, pH 7.0)
Separate solutions of disodium hydrogen phosphate in 100ml deionized water (25mM, by dissolving 0.445g) and sodium hydrogen phosphate monobasic in 100ml deionized water (25mM, by dissolving 0.39gm) are made and then pH 7.0 is maintained by adding two solutions slowly and measuring it by pH meter.
RNO Preparation (100 µM)
It is prepared by dissolving 0.15 mg in 10ml of 25mM sodium phosphate buffer. Wrap the bottle containing RNO solution with aluminium foil.
Dissolve 48.5mg in 25 ml of 25mM sodium phosphate buffer.
Sodium hypochlorite (100mM)
Commercially available sodium hypochlorite (Sigma) has molarity of 0.7M. We use 100mM, so add 28.5µl in the 200µl reaction system.
Hydrogen Peroxide (100mM)
Commercially available H2O2 has 9.7Molarity. We prepare 1M stock solution from which we add 20µl for 200µl reaction mixture.
Positive control (Rose Bengal) and negative control (DABCO) is prepared in deionized water likewise other samples either a crude extract from plant, cell, tissue, pure samples can be prepared in their respective solvent i.e. DMSO, ethanol or water.
Mark 96 well plate in such a way that one row in triplicate is used for L-histidine followed by another row in triplicate without L-histidine. Rows with L-Histidine contain 25µl L-histidine solution while rows without L-histidine contains same amount of buffer.
Add the test sample/standard in triplicate in both marked row of 96-well plate and keep control wells without any sample or standard.
For a standard sample, we used 100µM, 50µM, 25µM concentrations in triplicate in both L-histidine and without L-histidine rows.
For control wells (without any sample) we have added 106.5µl of 25mM sodium phosphate buffer for 200µl reaction mixture.
Add 20µl RNO in each well and keep the plate in a dark place.
CRITICAL STEP: wrap the RNO tube with aluminum foil because it is light sensitive.
- Then, add 20µl of 100mM H2O2 in each well followed by immediate addition of 28.5µl of 100mM NaOCl.
CRITICAL STEP: Add NaOCl immediately after H2O2 (there should be no time gap) otherwise RNO dye will be bleached as it is sensitive to hypochlorous acid and this may give false positive results.
Cover the 96-well plate with lid and wrap with aluminum foil then incubate the plate in the dark for 10 min at room temperature.
Finally, take the absorbance or kinetic reading (for 10 min) at 440nm in a microplate reader. Kinetic reading taken for 10 min with 5 sec orbital shaking, medium and read at 30-sec interval.
The graph is drawn with respect to control. Bleaching of RNO decreases the intensity of yellow color with decrease in optical density which indicates that singlet oxygen has not been quenched by the sample otherwise if there is no bleaching, it means that singlet oxygen has been quenched by the sample.
Percentage of singlet oxygen inhibition by sample is calculated by
= \(control O.D-sample O.D) X100
IC50 values can be derived using curve-fitting methods with statistical analysis.