G Protein Coupled Receptors represent one of the largest classes of cell surface receptors and one of the most important classes for drug targets. Fifty of the top 200 drugs target GPCRs. GPCRs respond to various stimuli like light, odors, hormones, neurotransmitters and others. They cover virtually all therapeutic areas. When a particular GPCR is implicated in a disease, researchers screen the GPCR and its signaling pathways, the hope being that promising therapeutic targets might be identified. Major G-protein families signal via secondary messengers like cAMP, which in turn activate a range of effector systems to change cell behavior and/or gene transcription. There are various approaches and methods to study GPCRs and measure the increase or decrease of intracellular cAMP. However, the fastest and the most sensitive among all methods is a plate based cAMP-Glo™ Assay.
While experienced researchers use this assay with ease, a beginner faces several obstacles including the uncertainty of where and how to start their testing. Like with any other biological assay one has to establish the best assay conditions. Here are some practical tips for HEK293 cell culture that can help the inexperienced user to determine the optimal cell number to use. Having additional reagents and more than one kit is good practice because one can make mistakes when preparing buffers.
Practical advice for seeding the cells
Start preparations one week in advance. For HEK293 cells, 100% confluency is approximately 10–12 million/75cm2 flask. Seeding Suggestion: Seed 1 million cells (from passage) in 15 ml DMEM + 10% FBS on a Friday so as to have 90% confluency after the weekend.
On Monday trypsinize cells and dilute to a density of 1 million cells/ml. Set up four flasks of cell culture:
- The first flask is used to test cell densities with Forskolin and to determine the best concentration of Forskolin. This flask is going to be used on Tuesday. Seed a sufficient number of cells in order to reach 80-90 % confluency overnight. This can be achieved by adding 4ml of HEK293 cells at a density of 1 million cells/ml to 11 ml DMEM + 10% FBS.
- The second flask will be used for the actual experiment (treatment) with the agonist and antagonist treatments. This flask is going to be used on Wednesday. Add 2.5ml of 1 million cells/ml of HEK293 cells into 12.5 ml DMEM + 10% FBS.
- The third flask is a reserve flask. Mix 1.5 ml of 1million cells/ml HEK293 cells into 13.5 ml DMEM + 10% FBS.
- The forth flask is used for passage of cells on Friday for the following Monday. Add 0.75ml of cells at a density of 1 million cells/ml to 14.25 ml DMEM + 10% FBS.
Optimization of cell numbers
Usually researchers test 3 cell densities. If one searches through the literature, there will be experiments performed with as little as 1,000 cells/well and as many as 50,000 cells/ well. What determines the range of cell numbers to be tested and how to find an optimal cell number?
The type and number of receptors as well as the cell type are the most important determinants. If your cells have natural receptors, and you are working in a 96-well plate, you should start with 10k, 25k or 50k cells/well. Primary cells often require higher numbers. If your cells are transfected with a specific receptor under a CMV or other strong promoter, you should begin by testing 1,000, 2,000 or 5,000 cells/well.
Some researchers choose to work with adherent cells, others work with cells that have been frozen or suspension cultures. All three kinds of culture influence the cell number to be used with cAMP-Glo™ Assay. The suggested HEK293 cell numbers are provided in Tables 1-3 of the Technical Bulletin for cAMP-Glo™ Assay TB357. Adherent cells are incubated overnight to allow the cells to attach to the growth matrix prior to the cAMP-Glo™ Assay. Suspension cells and frozen cells do not require an overnight incubation and can be used in the assay on the same day that they are prepared. When preparing suspension cultures we usually treat cells with Trypsin just prior to the assay. Here attention is needed because too long a Trypsin treatment can destroy receptors. Therefore do not exceed a treatment time of 1 minute with Trypsin.
The size of the well and plate are also important; in the Technical Bulletin for cAMP-Glo™ Assay TB357 three different ranges of HEK293 cell densities are given: for 96-well, 384-well or 1536-well plate.
Finding the best Forskolin Concentration
Once a researcher decides on the range of cell numbers to be tested, various concentration of Forskolin can be applied. Forskolin is a positive control that causes cells to make c AMP Glo independently of receptors. Therefore it is used to optimize cell numbers. As described in the above chapter, each cell type will have a different capacity for making cAMP and only the optimal concentration of Forskolin will give the highest response. For that reason we test various concentrations of Forskolin. There are several approaches when searching for optimal concentrations. The most practical approach is to start with a log scale. This is well presented in Figure 6 ( TB357). In this case data analysis was performed with GraphPad Prism® software, version 4.02, for Windows® using a sigmoidal dose-response (variable slope) equation. Once EC50 is determined, a researcher can perform two fold serial dilutions around EC50 as another way of optimizing the concentration.
The optimal Forskolin concentration is the one that gives the highest ratio when one divides the Luminescence (RLU) value obtained by background.
Once cell numbers and Forskolin concentrations are determined, agonist and antagonists are tested in an analogous manner. Use a known agonist or antagonist to determine the optimal conditions that result in the largest effect on cAMP levels.
I hope the above information will be useful. If I can offer any additional assistance, please leave a comment or contact Promega technical services.