Microplate multi reader infinite m200 pro

KI quenching assay was conducted in the presence and absence of ctDNA (60 μM). Briefly, AK-I-191 (20 μM) was dissolved in 10 mM Tris-HCl (pH 7.2) and titrated with increasing concentrations of KI. AK-I-191 was excited at 360 nm and fluorescence emitted at 420 nm was recorded. Fluorescence of AK-I-191 quenched by increasing concentrations of KI was also assessed without ctDNA. Quenching constant (Ksv) values in the presence and absence of ctDNA was analyzed via the Stern-Volmer equation. The fluorescence was measured using Microplate Multi-Reader Infinite M200 PRO (Tecan).

Prior to combined effects on anti-proliferative activity, cellular growth was assessed using WST reagent (EZ-Cytox, DoGenBio, Seoul, Korea). After 4 h starvation, compounds at the indicated concentrations were treated and then the cellular viability was measured using WST reagent and Microplate Multi-Reader Infinite M200 PRO (Tecan).
Combination index (CI) values were determined for drug-drug concentrations and effect levels (Fa, fraction affected; inhibition of cellular growth). CI values were calculated according to method of isobologram-combination index (Chou et al.) with CompuSyn software (ComboSyn Inc., Paramus, NJ, USA).

Hoechst 33258 (Hoechst), well-known as a DNA minor groove binder, was used to decipher the binding mode of compound on interaction with DNA. ctDNA (30 μM) and Hoechst (30 μM) were dissolved in 10 mM Tris-HCl (pH 7.2). Later, increasing concentrations of AK-I-191 were added to Hoechst-DNA solution. Solution was excited at 360 nm and emission spectra were recorded in the range 400-600 nm. Quenching constant (Ksv) values of AK-I-191 in reduction of Hoechst fluorescence was analyzed through the Stern-Volmer equation. The fluorescence was measured using Microplate Multi-Reader Infinite M200 PRO (Tecan, Männnedorf, Switzerland).