I am doing cAMP assay, using three ligands to study their effect on GPCR. I need to prepare 0.5 mM IBMX in stimulation buffer, and mixing it with cell suspention. Is there any specific method yo do this?
Hello Aisha Abdelmalik
The stimulation buffer should contain the test compound of interest along with 0.5mM IBMX. Stimulation buffer may be prepared using DPBS with Ca2+/Mg2+ with or without 0.1% BSA. If the stimulation is going to last for more than 2 hours, then you may have to use cell culture media instead of DPBS as the stimulation buffer containing the test compound of interest along with 0.5mM IBMX since this will help to limit the stress induced in cells.
If you are working with adherent cells, you may initially grow them in flask or plate. Then wash them with PBS and trypsinize. Resuspend the cell pellet in medium, and count the number of cells using a hemocytometer. Dispense the appropriate volume containing the recommended number of cells into each well of the assay plate, and allow the cells to incubate overnight. On the second day, remove the medium, and add the stimulation buffer containing the test compound of interest (1X) along with 0.5mM IBMX to the cells for an appropriate time to initiate induction.
If you are working with suspension cells, grow the cells in flask or plate. Centrifuge the cells and resuspend the cell pellet in stimulation buffer (without test compound and IBMX), and count the number of cells using a hemocytometer. Dispense the appropriate volume containing the recommended number of cells into each well of the assay plate. Add 2X of the test compound of interest along with 1mM IBMX in stimulation buffer and add to the cells which are already suspended in stimulation buffer to initiate induction for an appropriate time.
Please note that the cell density should be determined during assay development in cell titration experiments such that the measured signal falls within the linear range of the cAMP standard curve. If the cell density is high, there may be too many receptors present as a result of which it may reduce the effective free concentration of ligands and cause the assay to bottom-out.
Best.
If this is just a solubility question, you can dissolve IBMX in DMSO at room temperature. You could make a concentrated stock like this and store at -20 for later use. I usually prepare a small volume of 100mM IBMX in DMSO, then take whatever is needed to achieve 0.5mM in my buffer.
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