Coelenterazine

Protocol for Coelenterazine use^[1-3]

This protocol provides only a guide. Please modify according to specific needs.

Preparation of staining solution:

(1) Dissolve Coelenterazine in methanol to prepare a 4.7mM stock solution. Note: Aliquot unused stock solution and store at -20°C or -80°C protected from light. Avoid repeated freeze-thaw cycles.

(2) Dilute the stock solution with 0.1M potassium phosphate buffer to prepare a 0.4μM to 2.5μM working solution. Note: Adjust the working concentration according to actual conditions. Prepare fresh before each use.

Mitochondrial reactive oxygen species detection (using rat heart mitochondria as an example):

(1) Isolate rat heart mitochondria or submitochondrial particles (SMP). Adjust protein concentration to approximately 5mg/ml.

(2) Add 1 mg of mitochondria or SMP suspension to 2.5μM Coelenterazine and 2mg/ml NADH. Immediately measure cumulative luminescence signal within 15 seconds.

Cell staining:

(1) Culture cells expressing Renilla luciferase. Wash cells once with colorless solution.

(2) Add 0.4μM Coelenterazine. Incubate immediately at 37°C protected from light for 6 hours.

(3) Acquire images using a bioluminescence imaging system.

In vivo mouse imaging:

(1) Inject 2mg/kg Coelenterazine via tail vein into mice bearing xenograft tumors stably expressing Renilla luciferase.

(2) Allow Coelenterazine to circulate in vivo for approximately 5 minutes. Use a bioluminescence imaging system to capture images of orthotopic tumors and metastases. After sacrifice, homogenize major organs. Mix 50μL Coelenterazine substrate with 10μL tissue lysate. Quantify using a luminometer.

Precautions:

(1) Animal experiments must comply with animal welfare guidelines.

(2) For your safety and health, please wear a lab coat and disposable gloves during operation.

References:

[1] Volk-Draper LD, Rajput S, Hall KL, et al. Novel model for basaloid triple-negative breast cancer: behavior in vivo and response to therapy. Neoplasia. 2012 Oct;14(10):926-42.

[2] Raha S, Myint AT, Johnstone L, et al. Control of oxygen free radical formation from mitochondrial complex I: roles for protein kinase A and pyruvate dehydrogenase kinase. Free Radical Biology & Medicine. 2002;32(5):421-30.

[3] Pichler A, Zelcer N, Prior JL, et al. In vivo RNA interference-mediated ablation of MDR1 P-glycoprotein. Clin Cancer Res. 2005 Jun 15;11(12):4487-94.