STB-2

Operating Instructions
(The following is a recommended experimental protocol for guidance only, and needs to be adjusted according to your specific requirements).

1. Stock Solution Preparation
1.1 Solvent: Tetrahydrofuran (THF).
1.2 Concentration Recommendation: It is recommended to prepare a high-concentration stock solution of 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: Serum-free medium or PBS is usually used. Proteins and esterases in serum may interfere with staining results or cause dye hydrolysis.
2.2 Working Concentration: 50 μg/mL.
2.3 Notes: The working solution concentration can be adjusted as needed; prepare and use it immediately.

3. Staining Procedure
3.1 Sample Type^[1]:
3.1.1 Adherent cells: GL261 glioma cells.
3.2 Incubation Conditions: Incubate cells with 50 μg/mL STB-2@ApoE NPs for up to 8 h.
3.3 Washing Step: No washing step is specified.

4. Control Setup
4.1 Set up only when clearly mentioned in the literature; otherwise, this entire section can be omitted.
4.2 Set up negative controls, including PBS, PBS combined with 808 nm laser irradiation, STB-2 NPs without laser irradiation, and STB-2@ApoE NPs without laser irradiation.
4.3 Positive controls are used to verify the experimental system; blank controls are used to eliminate fluorescent interference from reagents.

5. Detection and Analysis
5.1 Instruments:
5.1.1 NIR-II fluorescence imaging system with an excitation wavelength of 808 nm, using long-pass filters (900 nm, 1000 nm, 1300 nm, 1400 nm, 1500 nm) to detect emitted light.
5.1.2 Two-photon fluorescence microscope with an excitation wavelength of 1700 nm, used for detecting NIR-II fluorescence.
5.1.3 Fluorescence microscope, used for intracellular ROS detection, with excitation of DCFH-DA (green fluorescence) and DAPI (blue fluorescence).
5.1.4 Flow cytometer, used for Annexin V-FITC/PI staining analysis.
5.2 Result Analysis:
5.2.1 Changes in fluorescence intensity: The enhancement of DCFH-DA green fluorescence indicates that 808 nm laser irradiation can induce ROS generation. After administration of STB-2@ApoE NPs, the NIR-II fluorescence intensity in the brain tumor region increases, reaching a peak at 12 h post-injection.
5.2.2 Fluorescence localization: STB-2@ApoE NPs accumulate in GL261 cells and orthotopic gliomas. The NIR-II fluorescence colocalizes with the bioluminescent signal produced by luciferase-labeled GL261 cells.
5.2.3 Changes in fluorescence color: DCFH-DA emits green fluorescence (ROS), DAPI emits blue fluorescence (cell nucleus), calcein-AM emits green fluorescence (live cells), PI emits red fluorescence (dead cells), and STB-2 NPs/STB-2@ApoE NPs emit NIR-II fluorescence (1000-1700 nm).

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.