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The problems with white plates are related to cross-talk blocking, surface reflectivity and tendency to phosphorescence. We do not recommend using white plates because they give much higher background even if adapted to darkness for 10 minutes. The white plastic does not block the light completely, and some portion of the signal will go through the plastic and will be seen when you measure adjacent wells. The typical situation is if you change from black to white plates, where the background is about doubled and the signal is increased about 10 times. Black plates are recommend for best signal to noise ratio even though the RLU values will be lower.
For Fetal Bovine Serum (FBS), there is no difference between 10% and 5% FBS. It is worthwhile to note that the type of serum does affect the luciferase assay. We tested the effect of 6 different types of sera (FBS, heat-inactivated FBS, dialyzed FBS, charcoal-treated FBS, donor adult bovine serum, and donor equine serum) on secreted luciferases, e.g. Gaussia, Gaussia-Dura, and Cypridina Luciferases. No noticeable difference was observed among different types of sera except for donor adult bovine serum. Unknown factors in donor adult bovine serum caused up to 35% inhibition in secreted luciferase activity. Therefore, we don’t recommend using donor adult bovine serum for Gaussia, Gaussia-Dura, and Cypridina Luciferases.
All Luciferase Glow Assay Working Solutions should be stored protected from light.
Here are possible causes and solutions:
Causes | Solution
|
Low transfection efficiency
| Optimize transfection conditions using a visual transfection control (e.g., a plasmid over-expressing a fluorescent protein) Verify plasmid DNA quality; use only transfection grade DNA Use actively dividing, low passage cells Use a different cell type |
No or low promoter activity
| Use conditions known for promoter activation Incubate cells for a longer time Change growth conditions to improve expression Use a different promoter |
Substrate auto-oxidized
| Protect substrate from light and air Maintain 100X Coelenterazine at -80°C; maintain 100X Vargulin at -80°C Prepare new Coelenterazine Working Solution if used longer than 8 hours; prepare new Vargulin Working Solution if used longer than 2 hours |
Here are possible causes and solutions:
Causes | Solution |
| Insufficient luciferase accumulation in media | Incubate cells for a longer time |
| Low luciferase expression | Use less media per well during the experiment Use a different promoter or growth conditions to improve expression Increase the integration time on the instrument Scale-up the volume of sample and reagent per well |
| Treatment interfered with cellular secretory pathway | Transfect cells with a plasmid for constitutive expression of Luciferase (i.e., with pTK or pCMV promoter); determine if luciferase actively expresses in media without treatment. Add treatment; determine if there is a corresponding drop in luciferase activity from the constitutively expressed plasmid |
Here are possible causes and solutions:
Causes | Solution |
| Non-optimized lysis buffer used | Assay luciferase activity in the media to confirm good expression of luciferase Use only the provided lysis buffer |
| Low luciferase expression | Lyse cells in smaller volume of 1X Cell Lysis Buffer Use a different promoter or growth conditions to improve expression Increase the integration time on the instrument Scale up volume of sample and reagent per well |
This could be due to high luciferase expression. Here are some suggestions:
Note: A low sample volume can increase assay variability. Dilute the sample and use the recommended volume of 10-20 μL per assay.
Here are possible causes and solutions:
Causes | Solution |
| Non-specific oxidation of substrate | Use less serum in the cell culture media Note: Albumin can increase the auto-oxidation of Vargulin Avoid repeated freezing and thawing of the sample |
| Control sample is contaminated | Use new sample Change pipette tips after each well |
Here are possible causes and solutions:
Causes | Solution |
| Low transfection efficiency | Optimize transfection conditions using a visual transfection control (e.g., a plasmid over-expressing a fluorescent protein) Verify plasmid DNA quality; use only transfection grade DNA Use actively dividing, low passage cells Use a different cell type |
| No or low promoter activity | Use conditions known for promoter activation Incubate cells for a longer time Change growth conditions to improve expression Use a different promoter |
| D-luciferin auto-oxidized | Protect substrate from light and maintain 100X D-luciferin at -20°C Prepare new Working Solution if used longer than 4 hours |
| Low luciferase expression | Use Pierce Firefly Signal Enhancer (100X) (Cat. No. 16180) Lyse cells in a smaller volume of 1X Cell Lysis Buffer Use a different promoter or growth conditions to improve expression Increase the integration time on the instrument Scale-up the volume of sample and reagent per well |
| Degraded luciferase protein | Store cell lysates on ice and perform assays immediately following cell lysis |
This could be due to high luciferase expression. Here are some suggestions:
This could be due to contamination of the control sample. Make sure to use new sample and change pipette tips after each well.
Here are possible causes and solutions:
Causes | Solution |
| Low transfection efficiency | Optimize transfection conditions using a visual transfection control (e.g., a plasmid over-expressing a fluorescent protein) Verify plasmid DNA quality using restriction digestion and agarose gel electrophoresis; use only transfection grade DNA Note: Most high-quality plasmid DNA should be supercoiled Use actively dividing, low-passage cells Use a different cell type |
| No promoter induction | Incubate cells using promoter-specific inducing conditions Incubate cells for a longer time after treatment Change growth conditions to improve expression Use a different promoter |
| Substrate auto-oxidized | Protect substrate from light and air Maintain 100X Coelenterazine at -80°C, 100X Vargulin at -20°C, and 100X D-Luciferin at -20°C, |
Here is a possible cause and its solutions:
Cause | Solutions |
| Low luciferase expression | Lyse cells in smaller volume of 1X Cell Lysis Buffer Use a different promoter or growth conditions to improve expression Increase the integration time on the instrument Scale up volume of sample and reagent per well |
This could be due to high luciferase expression. Here are some suggestions:
Here are possible causes and solutions:
Causes | Solution |
Non-specific oxidation of substrate | Use new sample Avoid repeated freezing and thawing of the sample |
Control sample is contaminated | Change pipette tips after each well Reduce shaker speed during the cell lysis step to avoid contaminating the wells |
Here are possible causes and solutions:
Causes | Solution |
| Low transfection efficiency | Optimize transfection conditions using a visual transfection control (e.g., a plasmid over-expressing a fluorescent protein) Verify plasmid DNA quality using restriction digestion and agarose gel electrophoresis; use only transfection grade DNA Note: Most high-quality plasmid DNA should be supercoiled Use actively dividing, low passage cells Use a different cell type |
| No promoter induction | Incubate cells under promoter-specific inducing conditions Incubate the cells for a longer time after treatment Change growth conditions to improve expression Use a different promoter or cell type |
| TurboLuc™ One-Step Substrate auto-oxidized | Protect substrate from light and air and store at -80°C Prepare Working Solution immediately before use and protect from light |
Wrong substrate used | Use only substrate supplied with the kit. Coelenterazine from related Luciferase kits (e.g., Renilla Luciferase) will provide suboptimal performance |
Low luciferase expression | Use a different promoter or growth conditions to improve expression Adjust instrument parameters to capture more signal (instrument-dependent) Plate larger number of cells |
Here are possible causes and solutions:
Causes | Solution |
| High luciferase expression | Reduce incubation time before collecting samples Adjust instrument parameters to capture less signal (instrument-dependent) Decrease the amount of plasmid transfected into cells or decrease cell number |
| Control sample contaminated | Change pipette tips after each well |
Using the Assay Stop Solution is optional, however the absorbance needs to be read immediately on the plate reader after the 30 minute incubation when the Stop Solution is not added to the assay, due to a shift in absorbance over time.