PreparationofLuciferinforInVitroandInVivoBioluminescentAssays

Preparation of Luciferin for
In Vitro Bioluminescent Assays
Materials
? D-Luciferin Firefly, potassium salt, 1.0 g /vial
(Caliper Life Sciences Part Number #119222)
? Sterile water
? Complete media
Procedure
1. Prepare a 200X Luciferin stock solution (30 mg/ml) in sterile water. Mix gently by inversion until Luciferin is completely dissolved. Use immediately, or aliquot and freeze at -20 °C for future use.
Note: One can either reconstitute the entire 1.0 g of D- Luciferin in 33.3 mL of sterile water to make the 30 mg/mL (200x) stock solution, or reconstitute the quantity of D-Luciferin necessary for an individual experiment.
2. Prepare a 150 μg/mL working solution of D-Luciferin in pre-warmed tissue culture medium.
Quick thaw 200X stock solution of Luciferin and dilute 1:200 in complete media (150 μg/mL final).
3. Aspirate media from cultured cells.
4. Add 1x Luciferin solution to cells just prior to imaging.
Note: Incubating the cells for a short time at 37 °C before imaging can increase the signal.
Preparation of Luciferin for
In Vivo Bioluminescent Assays
Materials
? D-Luciferin, Firefly, potassium salt, 1.0 g/vial
(Caliper Life Sciences Part Number #119222)
? DPBS, w/o Mg2+ and Ca2+
? Syringe filter, 0.2 um
Procedure
1. Prepare a fresh stock solution of Luciferin at 15mg/mL in DPBS. Filter sterilize through a 0.2 um filter.
2. Inject 10 μL/g of body weight. Each mouse should receive 150 mg Luciferin/kg body weight. (e.g. For a 10 g mouse, inject 100 μL to deliver 1.5 mg of Luciferin.)
3. Inject the Luciferin intra-peritoneally (i.p.) 10-15 minutes before imaging*.
* A Luciferin kinetic study should be performed for each animal model to determine peak signal time.