5× Firefly Lysis Buffer is a concentrated cell lysis buffer that is designed for use with Firefly Luciferase Assay Kit (F598310 and F773892).These kits are supplied with sufficient 5X Firefly Lysis Buffer to perform the stated number of assays with cells grown in 96 – 24 well plates. 5× Firefly Lysis Buffer is sold separately for use with the kits when more lysis buffer (e.g. >100 uL/well) is required.
Note: 5× Firefly Lysis Buffer is not compatible with Renilla Luciferase. If you require additional lysis buffer for use with Renilla Luciferase Assay Kit (R598308) and Firefly and Renilla Luciferase Assay Kit (D829607), order 5× Passive Lysis Buffer (P1375510).
Protocol
Preparation of cell lysates
1. Prepare 1× lysis buffer by adding 1 volume of 5× Firefly Luciferase Lysis Buffer to 4 volumes of dH2O and mixing well. 1× lysis buffer may be stored at 4°C for up to one month.
Lysis of Cells Cultured in Multiwell Plates
1. Remove growth medium from cultured cells and gently add a sufficient volume of phosphate buffered saline (PBS) to wash the surface of the culture vessel. Add 1× lysis buffer to each well using the volume recommended below for each type of culture plate:
| Wells/plate | Lysis buffer/well |
|---|
| 6 well | 500 uL | | 12 well | 250 uL | | 24 well | 100 uL | | 48 well | 65 uL | | 96 well | 20 uL |
|
2. Place the culture plates on a rocking platform or orbital shaker with gentle rocking/shaking to ensure complete and even coverage of the cell monolayer with 1× lysis buffer. Rock the culture plates at room temperature for 15 minutes. Note: Cultures that are overgrown are often more resistant to complete lysis and typically require an increased volume of lysis buffer and/or an extended treatment period to ensure complete lysis. Lifting cells from the plate will facilitate the process of cell lysis.
3. Transfer the lysate to a tube or vial. Optional: the lysate can be cleared by centrifugation for 30 seconds at top speed in a refrigerated microcentrifuge and transferred into a new tube. Place at 4°C for until ready to assay. Store lysates at -20°C or -70°C if assay will not be performed on the same day.
4. To assay lysates with Firefly Luciferase Assay Kit (F598310 or F773892), follow the assay protocols provided with the kit.
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