Preparation of Luciferin for In Vitro and In Vivo Bioluminescent Assays

实验概要

Reagent  for immunoassay, ligand binding assay and ligand receptor assay in  which luciferin is covalently bonded to a molecule having biological  activity for bonding to a particular biologically active group of a  material and method in which the luciferin and biologically active  molecule conjugate is added to a material having a group for combination  with the biological activity of the conjugated molecule, the mixture is  incubated to bond the material to the reagent through the biological  activity and the product of the bonding is reacted with luciferase to  produce bioluminescence having intensity dependent on the concentration  of the material being assayed.

主要试剂

D-Luciferin Firefly, potassium salt, 1.0 g /vial [Detail: Caliper Life Sciences Cat#119222]

Sterile water

Complete media
DPBS, w/o Mg² and Ca²  

实验步骤

Preparation of Luciferin for In Vitro Bioluminescent Assays

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 (150μg/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

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.