AF633 C5 Maleimide

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

1. Stock Solution Preparation
1.1 Solvent: Acetonitrile.
1.2 Concentration recommendation: 25 μM; alternative: 1 mg per 500 μL.

2. Working Solution Preparation
2.1 Diluent: 1:1 mixture of acetonitrile and water plus phosphate buffer solution (PBS) pH 5.7.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type descriptions
3.1.1 For extracellular vesicles (EVs)[1]: EVs at 3.7 mg/mL concentration.
3.1.2 For purified Nestin (Nes) peptide[2]: Purified peptide sample.
3.2 Incubation conditions
3.2.1 For extracellular vesicles (EVs)[1]: Incubate with AF633 C5 Maleimide overnight at 4°C.
3.2.2 For purified Nestin (Nes) peptide[2]: Incubate in the dark for 30 min at room temperature (21 °C).
3.3 Washing steps
3.3.1 For extracellular vesicles (EVs)[1]: Remove dye remnants by passing the stained EV sample through an Exo Spin column.
3.3.2 For purified Nestin (Nes) peptide[2]: No washing steps specified; functionalized peptide is purified via preparative reversed phase HPLC after incubation.

4. Controls
4.1 Filtered PBS (procedural control) for single vesicle flow cytometry assays.
4.2 Unsorted cell population and complete cell library control for flow cytometry EV uptake assays.

5. Detection & Analysis
5.1 Instrument type: Flow cytometer, confocal fluorescence microscope, electrospray ionization mass spectrometer (ESI-MS), confocal laser scanning microscopy (cLSM).
5.2 Ex/Em wavelengths: Alexa 633 fluorophore-specific excitation/emission wavelengths.
5.3 Result analysis
5.3.1 For extracellular vesicles (EVs)[1]: Cells with reduced EV uptake show lower AF633 fluorescence, while cells with enhanced EV uptake show higher AF633 fluorescence; AF633 fluorescence co-localizes with EV markers CD81, CD9, CD63, and LAMP2; red fluorescence is emitted from the stained EVs and internalized EVs.
5.3.2 For purified Nestin (Nes) peptide[2]: ESI-MS confirms purity of the AF633 C5 Maleimide-labeled peptide; the labeled peptide-functionalized gold nanorods exhibit red fluorescence, accumulate uniformly throughout glioblastoma multicellular tumor spheroids (MCTS), localize inside endosomes, and show increased mean fluorescence intensity with prolonged incubation time indicating enhanced cell uptake.

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