8pyDTZ

The use of 8pyDTZ in HEK 293T cells to evaluate its bioluminescence performance：
1. Culture HEK 293T cells in DMEM with 10% FBS at 37°C with 5% CO₂.
2. Perfom transfection when cell confluency reaches 70-80%.
3. Transiently transfect cells with the LumiLuc luciferase expression plasmid using liposome-based transfection
4. The expression of LumiLuc luciferase in the cells was detected 24-48 hours after transfection.
5. Dissolve 8pyDTZ in ethanol to a concentration of 10mM.
6. Use an assay buffer composed of 1mM CDTA, 0.5% Tergitol NP-40, 0.05% Antifoam 204, 150mM KCl, 100mM MES pH 6.0, 1mM DTT, and 35mM thiourea.
7. Dilute the 8pyDTZ stock solution (10mM) in the assay buffer to final concentration of 20µM.
8. Seed transfected cells into 96-well plate at a density of 5,000 cells/well.
9. Allow cells to adhere and recover for 2-4 hours in the incubator.
10. Add 50µL of the 8pyDTZ (20µM) containing solution to each well containing the transfected cells.
11. Incubate the cells with 8pyDTZ at 37°C for 10 minutes.
12. Perform bioluminescence imaging immediately after incubation using a luminescence plate reader with exposure time of 2s.
13. Use a 600-700nm bandpass filter to capture far-red emission.
This protocol only provides a guideline, and should be modified according to your specific needs.

References:
[1] Yeh H W, Xiong Y, Wu T C, et al. ATP-Independent Bioluminescent Reporter Variants To Improve in Vivo Imaging. ACS Chem Biol. 2019 May 17;14(5):959-965.