Protocol I (Luminometers without injectors) (E3309)
- Thaw BioLux Cypridina Luciferase Assay Buffer (1X) completely to room temperature (protect from light) and mix well before use.
- Prepare the CLuc assay solution (e.g. for 100 samples add 50 μl of the reconstituted substrate (100X solution) to 5 ml of BioLux Cypridina Luciferase Assay Buffer).
- Mix well by inverting the tube several times (do not vortex).
- Incubate at room temperature for 30 minutes (protect from light).
- Set the luminometer for 2–10 seconds of integration.
- Pipet samples *(5–20 μl per well) into a 96-well white (opaque), black plate or cuvette.
* Approximately 90% of Cypridina Luciferase is secreted into the culture medium after transfection and thus, the CLuc activity is typically assayed in the supernatant (i.e. culture medium of CLuc-transfected cells). However, as long as the cells are alive, ~10% of CLuc is present inside the cell and therefore, the CLuc activity can also be assayed in the cell lysate (Figure 7). We recommend that the cell lysates be prepared by using Luciferase Cell Lysis Buffer (#B3321), since this lysis buffer is designed to be compatible with Cypridina, Gaussia, Renilla, Firefly Luciferase and β-gal activity assays.
- Add the CLuc assay solution (50 μl) to a sample (i.e. add the assay solution to only one sample at a time) and promptly proceed with the measurement.
- Repeat Step 7 for all samples.