Nile-TPA

Operating Instructions
(The following is a recommended experimental protocol for guidance only; adjust according to specific requirements)^[1]


1. Preparation of Working Solution
1.1 Working concentration: 10-50 μM.
1.2 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

2. Staining Procedures
2.1 Sample types:
2.1.1 Adherent cells: HL-7702 cells, SiHa cells.
2.1.2 Zebrafish.
2.1.3 Mouse blood samples.
2.2 Incubation conditions:
2.2.1 Adherent cells: Incubate with 10 μM Nile-Tpa for 30 min.
2.2.2 Zebrafish control group: Incubate with 10 μM Nile-Tpa for 30 min; zebrafish experimental group: First incubate with LPS (HY-D1056) (1.6 mg/mL) for 30 min, then incubate with 10 μM Nile-Tpa for 30 min.
2.2.3 Mice: Inject 50 μM Nile-Tpa 1 hour before imaging.
2.3 Washing procedures:
2.3.1 Adherent cells: Rinse 3 times with PBS buffer.
2.3.2 Zebrafish: Rinse with PBS before imaging.

3. Control Settings
3.1 Cell imaging: Use normal HL-7702 cells as the control for cancerous SiHa cells.
3.2 Zebrafish imaging: Zebrafish incubated only with 10 μM Nile-Tpa serve as the control for zebrafish pretreated with LPS.
3.3 Mouse imaging: Use normal mice as the control for LPS-induced inflammatory mice; use mice injected with PBS via tail vein as the control for mice with circulating tumors.
3.4 Positive controls are used to validate the experimental system; blank controls are used to eliminate fluorescent interference from reagents.

4. Detection and Analysis
4.1 Instruments:
4.1.1 Inverted laser scanning confocal microscope: Use Ex = 561 nm, Em = 570-620 nm (TRICT channel) for cell and zebrafish imaging.
4.1.2 In vivo imaging system: Use Ex = 560 nm, Em = 620 nm for mouse imaging.
4.2 Result analysis:
4.2.1 Changes in fluorescence intensity:
4.2.1.1 The lower the solvent polarity, the higher the fluorescence intensity; the higher the solvent polarity, the lower the fluorescence intensity.
4.2.1.2 The fluorescence intensity of cancerous SiHa cells is higher than that of normal HL-7702 cells.
4.2.1.3 The fluorescence intensity of zebrafish pretreated with LPS is higher than that of control zebrafish.
4.2.1.4 The fluorescence intensity of LPS-induced inflammatory mice is higher than that of normal mice.
4.2.1.5 The fluorescence intensity of blood samples from mice with circulating tumors is higher than that of normal mice, indicating lower blood polarity in the former.
4.2.2 Fluorescence localization: Targets intracellular lipid droplets.
4.2.3 Color change: Emits red fluorescence in low-polarity environments.

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