Reporter assays introduce a reporter gene into cells and detect the product of the reporter gene when the gene is transcribed and translated by the cell. The reporter can include a protein tag or an enzyme such as luciferase. In this example, the LC3 protein, widely used as a marker of autophagic activity, is tagged with HiBiT, a small 11a.a. peptide tag. Cells expressing the Autophagy LC3 HiBiT Reporter are treated with inducers or inhibitors of autophagy. At the end of the experiment, cells are lysed and total LC3 is quantitated by adding the NanoGlo® HiBiT Lytic Detection Reagent, which includes the complementing LgBiT protein and NanoLuc® substrate.
The Autophagy LC3 HiBiT Reporter was introduced into HEK293 cells, and the reporter cells were grown in both monolayer and 3D spheroids. Does the LC3 HiBiT Reporter signal accurately reflect reporter levels in 3D spheroids?
Adaptation for 3D Models
In this assay, to detect and quantify the tagged protein, the cells must be fully lysed to recover the tagged protein from all the cells. In order to facilitate lysis of the 3D spheroids, the protocol was adapted to increase the shaking time and reagent processing time after addition of the lytic reagent. The 2-minute shaking plus 10-minute incubation for monolayers was increased to 30 minutes of spheroid shaking and incubation.
The performance of the Autophagy LC3 HiBiT Reporter Assay System in 3D models was verified by comparing the recovery of the reporter to the ATP content in the spheroid model, wherein ATP is a useful surrogate for cell number. Different size spheroids were created from the HEK293 reporter cells by seeding increasing cell number in Corning® Ultra-Low Attachment 96-well plates. After 4 days of culture, spheroids of a wide range of sizes were generated for subsequent assay of autophagy reporter levels with the NanoGlo® HiBiT Lytic Detection Reagent. In parallel plates, a similar set of spheroids was processed with CellTiter-Glo® 3D to determine ATP content.