Iraninan Biology Society Journal of Cellular and Molecular Research (Iranian Journal of Biology) 2383-2738 36 2 2023 06 22 The Role of Pre-incubation in Formation of Supramolecular Complex Between Lucigenin and p-Sulfonatocalix[4]arene in Cell Culture Medium The Role of Pre-incubation in Formation of Supramolecular Complex Between Lucigenin and p-Sulfonatocalix[4]arene in Cell Culture Medium 116 124 2150 FA Reihaneh Khosravi Bioprocess engineering department, national institute of genetic engineering and biotechnology (NIGEB) 0009-0000-2200-7607 Amir Norouzy Bioprocess engineering department, national institute of genetic engineering and biotechnology (NIGEB), Tehran, Iran Journal Article 2021 05 24 Lucigenin (LCG) is a fluorescent dye that can form supramolecular complex with p-Sulfonatoclix[4]arene by doing a simple incubation. By formation of CX4‧LCG complex, fluorescence intensity of LCG is quenched. The complex is used in indicator displacement assay (IDA), in which an analyte (A) is incubated with the CX4‧LCG complex, the A then displaces LCG and CX4‧A complex forms in real time. The liberated LCG regains its fluorescence. In cell culture medium there are ample of competitor ligand molecules for LCG. The presence of competitors decreases the association constant (ka) of LCG to CX4. In this study, the CX4‧LCG complex were pre-incubated with tetra-arginine peptide in phosphate buffer, a solution devoid of any competitor molecule, prior to dilution with the cell culture medium to the desired concentration. The fluorescence intensity of the solution was measured hoping for maximum fluorescence recovery. Next time, all incubations were performed in the medium then the fluorescence was measured. Contrary to popular belief, two experiments did not show a significant difference i.e., the ka value remains unchanged. We concluded that the supramolecular interactions will come to a multi-lateral equilibrium; therefore, IDA can be used for monitoring and measuring the concentration of analyte-of-interest even in the presence of competitors in the cell culture medium which is an interesting solvent in many biological sciences. Lucigenin (LCG) is a fluorescent dye that can form supramolecular complex with p-Sulfonatoclix[4]arene by doing a simple incubation. By formation of CX4‧LCG complex, fluorescence intensity of LCG is quenched. The complex is used in indicator displacement assay (IDA), in which an analyte (A) is incubated with the CX4‧LCG complex, the A then displaces LCG and CX4‧A complex forms in real time. The liberated LCG regains its fluorescence. In cell culture medium there are ample of competitor ligand molecules for LCG. The presence of competitors decreases the association constant (ka) of LCG to CX4. In this study, the CX4‧LCG complex were pre-incubated with tetra-arginine peptide in phosphate buffer, a solution devoid of any competitor molecule, prior to dilution with the cell culture medium to the desired concentration. The fluorescence intensity of the solution was measured hoping for maximum fluorescence recovery. Next time, all incubations were performed in the medium then the fluorescence was measured. Contrary to popular belief, two experiments did not show a significant difference i.e., the ka value remains unchanged. We concluded that the supramolecular interactions will come to a multi-lateral equilibrium; therefore, IDA can be used for monitoring and measuring the concentration of analyte-of-interest even in the presence of competitors in the cell culture medium which is an interesting solvent in many biological sciences. https://cell.ijbio.ir/article_2150_24917db15c4e37e421866448c9ab23d8.pdf