TPEP-6-NH-MA

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

1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Concentration recommendation: Prepare a stock solution such that dilution at 1:100 (v/v) with PBS yields a 1 μM working solution.

2. Working Solution Preparation
2.1 Diluent: PBS (phosphate buffered saline).
2.2 Working concentration: 1 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type: Adherent cells (H1299 non-small cell lung cancer cells).[1]
3.1.1 Adherent cells: No trypsinization specified for staining step; cells are cultured to adhere before staining.
3.2 Incubation conditions: Incubate cells with 1 μM probe working solution at 37°C for 1 h; for pre-treated control cells, incubate with 1 μM probe working solution at 37°C for 20 min.
3.3 Washing steps: Wash cells three times with PBS buffer before and after incubation.

4. Controls
4.1 Negative control: H1299 cells pretreated with 100 μM N-methylmaleimide in DMSO-PBS (5:100, v/v, pH 7) at 37°C for 1 h to eliminate intracellular free thiols, then incubated with 1 μM probe working solution.

5. Detection & Analysis
5.1 Instrument: Inverted fluorescence microscope.
5.1.1 Ex wavelength: 361 nm for TPEP-6-NH-MA; Em wavelength corresponding to blue fluorescence emission.
5.2 Result analysis:
5.2.1 Fluorescence intensity changes: Non-fluorescent probe exhibits strong fluorescence enhancement (over 2000-fold) upon reaction with Cys or Hcy; no significant fluorescence enhancement with GSH.
5.2.2 Color changes: Blue fluorescence is observed in cells with endogenous thiols after probe incubation; no blue fluorescence is observed in N-methylmaleimide-pretreated cells.
5.2.3 Fluorescence localization: Blue fluorescence is observed in the intracellular space of H1299 cells.

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