The Luciferase Assay System from Promega is available in two different formats. You can buy the system without Reporter Lysis Buffer or with Reporter Lysis Buffer. Whether you get the Reporter Lysis Buffer or not, you will receive vials of lyophilized luciferase substrate and luciferase assay buffer. If you get the Reporter Lysis Buffer, it comes as a 5x concentrated solution. The product is really simple to use. You simply combine the luciferase assay buffer with the lyophilized luciferase substrate and you have your substrate. When harvesting the cells, the Reporter Lysis Buffer is simply added to the cell culture you are using; you can freeze the cells to aid the lysis. After lysis of the cells, a small amount of this sample is added to a white OptiPlate (OptiPlate-96 White from PerkinElmer) with the luciferase substrate and is then easily read on a plate reader or luminometer with a luminescence optic. These white OptiPlates are not included in the kit and must be purchased separately. One thing to keep in mind is that the light emitted from the samples is short lived so I’d recommend adding substrate to and reading no more than 30 samples at a time.
I first utilized this system with a HeLa cell line stably transfected with a luciferase NfêB reporter. I transfected this cell line with multiple immune receptors to determine NfêB activation. It was easy to dilute the Reporter Lysis Buffer and add it to the cells after the media was taken off the cells. After freezing the cells at -80°C, I added the substrate and read the plate.
This product was great to use and easy to prepare. The lyophilized luciferase substrate was very easy to dissolve in the provided luciferase assay buffer. When reading the results in the plate reader, the results were consistent with what was expected and controls showed low to no luciferase activity. It gave a nice low background reading. The data that the luciferase system gave was simple to analyze and I was able to get reproducible results from my experiment. This system combined with my immune receptor-transfected HeLa cell line provided an easy way to look at NfêB activation and allowed me to determine which receptors are actually recognizing certain antigens.