D-Luciferin

1.Example protocol for in vitro bioluminescent image assays
a) Prepare a 100 mM (100-200X) Luciferin stock solution in sterile water. Mix well. Use immediately, or make single use aliquots, and store at -20℃, avoid freeze-thaw cycles, avoid exposure to the light.
b) Prepare a 0.5-1 mM working solution of D-Luciferin in pre-warmed tissue culture medium.
c) Aspirate media from cultured cells.
d) Add Luciferin working solution to cells, and incubate the cells for 5-10 minutes at 37℃ just prior to imaging.
2.Example protocol for in vivo bioluminescent image assays
a) Prepare a 15 mg/mL Luciferin stock solution in DPBS, without Mg2+ and Ca2+. Mix well.
b) Filter sterilizes the solution through a 0.2 μm filter. Use immediately, or make single use aliquots, and store at -20℃, avoid freeze-thaw cycles, avoid exposure to the light.
c) Inject the luciferin intra-peritoneally (i.p.) 10-15 minutes before imaging at 150 mg/kg (or 10 μL/g of luciferin stock solution) of the animal body weight.
Note: A kinetic study of luciferin should be performed for each animal model to determine peak signal time.
3.Example protocol for luciferin reporter assays
a) Prepare a 100 mM Luciferin stock solution in sterile water. Use immediately, or make single use aliquots, and store at -20℃, avoid freeze-thaw cycles, avoid exposure to the light.
b) Prepare a 1 mM working solution of D-Luciferin with 3 mM ATP, 1 mM DTT and 15 mM MgSO4 in 25 mM tricine buffer pH 7.8.
c) Pipette 5-10 μLof cell lysate into a microplate. Use lysis reagent or buffer without lysate as a blank.
d) Prime luminometer with luciferin working solution.
e) Inject 200 μL of luciferin working solution with no delay and a 10 second integration time.

In an immunocompetent mouse model of ovarian cancer, use of D-luciferin substrate and firefly luciferase preserves tumor-host immune interactions, since bioluminescent imaging is a more sensitive indication of tumor growth than weight gain. ^[2]