Adapted from manufacturers protocol

General description

Measures cytochrome P450 CYP activity–specifically CYP3A4.

A proluciferin substrate provided in the kit (Luciferin-PFBE) is acted upon by the CYP enzymes in the cultured cells to be tested to produce D-luciferin. Luciferin Detection Agent provided in the kit is added to the cells, which lyses them and reacts with the released D-luciferin to produce light. More light = more D-luciferin = more P450 activity.

Because the light detection needs to happen in plates with black or white sided wells (not clear), either the cells need to be cultured in those plates (preferred method) or the lysate + Luciferin Detection Agent needs to be transfered from a normal culture plate to a black or white plate before measuring luminescence, taking care to transfer equal volumes for each well. Promega recommends white plates for highest luminescence readings, but black work for the range of luminescence we are seeing.

A standard curve should be generated to quantify how much light is produced by different amounts of D-luciferin. Make multiple dilutions from a known quantity of D-luciferin (Beetle D-luciferin, NOT provided with the kit), and react them with the Luciferin Detection Agent using the same concentrations/volumes as the experimental wells.

A No-cell control and vehicle controls (if some cells have been treated with AFB in DMSO, for example) are needed.

This version of the protocol should only be used on cells. Non-cell-based assays need additional reagents to jump-start the proluciferin/P450 reaction.

Process

  1. Start with cells cultured in 96 well plates, treated as desired (e.g., control vs AFB) for 24-72 hours.
  2. Prepare Substrate Media.
  3. Remove media from cells and wash with media or PBS. Remove the media or PBS.
  4. Add 50uL Substrate Media to each cell well and control well.
  5. Incubate at 37C 5% CO2 for 3-4 hours.
  6. During incubation, prepare Luciferin Detection Reagent and Standard Dilutions.
  7. Add 50uL of appropriate Standard Dilution to each standard well.
  8. To each cell well, control well, and standard well add 50uL Luciferin Detection Reagent.
  9. Mix briefly on a plate shaker to form lysate.
  10. Read luminescence from plate using 1 seconds per plate integration time (Promega recommends 0.25-1 second). Do not use a fluorometer and do not use filters with the luminometer.

RECIPES

Substrate Media

50uM Luciferin-PFBE in Definitive Media

Per well of a 96 well plate (multiply by number of cell wells and control wells plus 5-10% for pipette loss):

  • 1.25uL Luciferin-PFBE (provided with kit)
  • 48.75uL Definitive Media

Standard Dilutions

Beetle D-luciferin Potassium Salt in Definitive Media at various concentrations:

Stock Beetle Luciferin solution (2mM in water)

  • 5mg Beetle Luciferin Potassium Salt
  • 7.85mL mqWater
  • Aliquot and store in -80 freezer.

Working Beetle Luciferin solution (80uM in media)

  • 20uL Stock Beetle Luciferin solution
  • 480uL Definitive Media
  • Use this Working Beetle Luciferin solution to make 0, 0.032, 0.16, 0.8, and 4uM standard dilutions in media (see setup sheet).

Luciferin Detection Reagent

  • Add entire bottle of reconstitution buffer (provided with kit) to amber bottle of lypholized Luciferin Detection Reagent (provided with kit).
  • Mix by swirling or inverting several times.
  • The reconstituted Luciferin Detection Reagent can be stored at room temperature for 24 hours or at 4C for 1 week without loss of activity. For long-term storage, store at –20°C for up to 3 months. Be sure to mix the thawed Luciferin Detection reagent well before use.