CDPP-2PF6

Guidelines (The following is our recommended protocol. This protocol is for reference only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: DMSO.

2. Working Solution Preparation
2.1 Diluent: serum-free medium or PBS.
2.2 Working concentration:
- Cell membrane staining/labeling: 10 μM CDPP-2PF6.
- Migrasome staining/imaging: 20 μM CDPP-2PF6.
- PIP2 lipid vesicle staining/labeling: 1-10 μM CDPP-2PF6.
2.3 Note: Adjust the working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 For adherent cells^[1]:
3.1.1 Cell lines:
- HeLa, H1299, L929-TSPAN4 (GFP), 4T1, Hep G2, RAW 264.7, 3T3, and H1299-TSPAN4 cells.
3.1.2 Cell membrane staining:
3.1.2.1 Incubate adherent cells with 10 μM CDPP-2PF6 at 37°C in the dark for 0-4 h.
3.1.3 Migrasome staining:
3.1.3.1 Incubate adherent cells with 20 μM CDPP-2PF6 for 5 to 120 min.
3.1.3.2 For migrasome co-localization, wash three times with PBS after co-incubation.

3.2 For PIP2 lipid vesicles:
3.2.1 Prepare PIP2-containing lipid vesicles. Take 500 μL of PIP2 lipid solution for labeling.
3.2.2 Incubate the vesicles with 10 μM CDPP-2PF6 for 20 min.

4. Controls
4.1 Co-staining controls:
4.1.1 DiO (HY-D0969) is used for cell membrane co-localization validation.
4.1.2 WGA or TSPAN4-GFP is used for migrasome co-localization validation.
4.1.3 Lipid-tracker Green is used for PIP2 vesicle co-localization validation.
4.2 Pharmacological controls: Blebbistatin (HY-13813) and Methyl-β-cyclodextrin (MβCD) (HY-101461) are used to validate lipid-dependent staining specificity.
4.3 Blank control: lipid-free blank control for fluorescence spectral analysis.

5. Detection and Analysis
5.1 Instrument type: fluorescence microscope; fluorescence spectrometer.
5.2 Wavelength settings: excitation wavelength (Ex) of CDPP-2PF6 is 450 nm; emission wavelength (Em) is approximately 760 nm.
5.3 Result analysis:
5.3.1 Fluorescence intensity changes: upon binding to cell membranes, migrasomes, and PIP2-containing vesicles, strong red fluorescence is enhanced; fluorescence intensity increases with increasing PIP2 concentration.
5.3.2 Fluorescence localization: selectively accumulates in cell membranes, migrasomes, retraction fibers, and PIP2-rich membrane domains; for cell membranes, it shows high co-localization with DiO, and for migrasomes, it shows partial co-localization with WGA/TSPAN4-GFP.
5.3.3 Color change: CDPP-2PF6 staining emits red fluorescence.
5.3.4 CDPP-2PF6 labels cell membranes and migrasomes and can be observed by confocal microscopy; PIP2 lipid vesicles are labeled with CDPP-2PF6 and imaged by confocal microscopy. Fluorescence lifetime imaging can be performed.

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