Golgi-hNR

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: DMSO.
1.2 Concentration recommendation: 1.0 mM.

2. Working Solution Preparation
2.1 Diluent:
- For spectral measurements: BR buffer (40 mM, acetic acid, phosphoric acid, and boric acid) containing 4.0% DMSO.
- For cell experiments/imaging: Dulbecco’s modified Eagle’s medium (DMEM).
2.2 Working concentration:
- For spectral measurements: 40 μM.
- For cell experiments/imaging: 3 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type descriptions: Adherent cells (HeLa, HepG-2, A549 cell lines)[1][2][3]:
3.1.1 No trypsinization is required for staining steps.
3.2 Incubation conditions:
3.2.1 Incubate cells with 3 μM Golgi-hNR (optional co-incubation with 500 nM Golgi-Tracker Green) at 37°C for 30 min[1][2].
3.2.2 Staining results in red fluorescence localization to the Golgi apparatus, with intensity decreasing as pH increases[3].
3.3 Washing steps: Wash cells with PBS buffer (pH 7.4) three times after incubation[1][2].

4. Detection & Analysis
5.1 Instrument type: Confocal laser scanning microscope[1][2]; fluorescence imaging instrument[3].
5.2 Ex/Em wavelengths:
5.2.1 For Golgi-hNR:
- Excitation: 560 nm[2], 561 nm[1][2].
- Emission: 615 nm[3], 568-650 nm[2], 663-738 nm (Cy5 channel)[1], 664 nm[2].
5.3 Result analysis:
5.3.1 Fluorescence intensity changes: Fluorescence intensity increases as pH decreases from 7.4 to 3.0; intensity decreases as pH increases, including under alkaline stimulation (e.g., NH4Cl, chloroquine), and can recover after removing NH4Cl[1][2][3].
5.3.2 Fluorescence localization: Red fluorescence of Golgi-hNR selectively localizes to the Golgi apparatus of HeLa, HepG-2, and A549 cells, with high co-localization confirmed by Pearson's and Mander's coefficients when paired with Golgi-Tracker Green[1][2][3].
5.3.3 Color changes: Emits red fluorescence under acidic conditions, with negligible fluorescence at neutral/alkaline pH[1][2][3].