At baseline, KB 3-1 Rluc tumors show a 4-fold higher bioluminescence signal in vivo than KB 8-5-11 Rluc tumors. Normalized bioluminescence signals arise from KB 3-1 Rluc tumors, signals emits from KB 8-5-11 Rluc tumors in vivo are consistently reduced by ≈75%[1] .
In Vitro
On addition of Coelenterazine to the buffer, intact KB 3-1 Rluc cells show high bioluminescence, whereas intact KB 8-5-11 Rluc cells show low bioluminescence. A specific Pgp-mediated reduction in steady-state content of Coelenterazine exists in KB 8-5-11 Rluc cells compare to KB 3-1 Rluc cells. Bioluminescence signals from KB 3-1 Rluc cells, both in the absence and presence of GF120918 (300 nM), are readily detected with as little as 1 nM extracellular Coelenterazine and increase in a concentration-dependent manner to 1 μM extracellular Coelenterazine. In Pgp-expressing cells, there is evidence for a GF120918-reversible concentration-dependent saturation of bioluminescence with an EC50 of 257 nM Coelenterazine. The plateau in bioluminescence signal observed in KB 8-5-11 Rluc cells implies hat the capacity of Pgp to limit delivery of Coelenterazine to cytosolic Rluc is not exceeded within the concentration range test[1].
Kinase Assay
Cell Assay
Cells are plated at a density of 5×104 cells per well into 24-well plates and grown to 80-100% confluency. Just before imaging, media are changed to a colorless solution containing (in mM): 2.7 KCl, 139 NaCl, 8.1 Na2HPO4, 0.7 H2O, 1.5 KH2PO4, 1.8 CaCl2, 1 MgCl2 and 5.5 d-glucose. Cells are preincubated for 15 min in the absence or presence of Pgp modulator, after which Coelenterazine (final concentration of 470 nM) is added directly to the cells[1].
Animal Administration
Mice are anesthetized with metofane or isoflurane before tail vein injection of Coelenterazine (4 μg/g) formulated from an ethanol stock diluted in sodium phosphate buffer (50 mM). Bioluminescence imaging is performed on the in vivo imaging system at 2, 6, 8, and 11 min after injection. Anesthesia is maintained during imaging by nose cone delivery of 2.5% isoflurane. After imaging, animals are killed by cervical dislocation; tumors are then harvested and weighed[1].
References
[1]. Andrea Pichler, et al. Imaging reversal of multidrug resistance in living mice with bioluminescence: MDR1 P-glycoprotein transports coelenterazine. Proc Natl Acad Sci U S A. 2004 Feb 10; 101(6): 1702–1707.
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