Disulfo-Cy3 NHS ester

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: 20 mM.

2. Working Solution Preparation
2.1 Diluent: Hanks’ balanced salt solution (HBSS); alternative diluent: FACS buffer (DPBS supplemented with 2% FBS).
2.2 Working concentration: 0.1 μM, 1 μM, 10 μg/mL, 20 μM.
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 adherent cells (U87, HeLa)[1]: Wash once with warm HBSS before staining; detach with StemPro Accutase for downstream flow cytometry or coculture experiments.
3.1.2 For suspension cells (NK-92 cells)[2]: Centrifugation is used for washing steps; incubate with IFN-γ at 37°C for 1 h prior to dye incubation.
3.2 Incubation conditions
3.2.1 For adherent cells[1]: Incubate with 0.1-1 μM disulfo-Cy3 NHS ester working solution for 10 min at room temperature.
3.2.2 For suspension cells[2]: Incubate with 10 μg/mL or 20 μM disulfo-Cy3 NHS ester working solution on ice for 30 min.
3.3 Washing steps
3.3.1 For adherent cells[1]: Wash twice with HBSS after incubation.
3.3.2 For suspension cells[2]: Wash three times with FACS buffer after IFN-γ incubation; wash three times with FACS buffer after dye incubation.

4. Controls
4.1 Unlabeled cells as negative control.
4.2 Cells incubated with 100 ng/mL IFN-γ without stimulation as positive control.
4.3 A549 cells (low IFN-γ secretion) as specificity control.

5. Detection & Analysis
5.1 Instrument type: Flow cytometer, confocal fluorescence microscope.
5.2 Ex/Em wavelengths
5.2.1 Flow cytometer: Excitation wavelength 561 nm, emission detection at 579-593 nm.
5.2.2 Confocal fluorescence microscope: Excitation wavelength 552 nm, emission detection at 570-620 nm.
5.3 Result analysis
5.3.1 Red fluorescence is observed for stained cells.
5.3.2 Fluorescence localizes to the cell membrane; adherent cells show weak plasma membrane staining with lower labeling density than membrane-anchored analogues.
5.3.3 Fluorescence intensity changes: Adherent cells show lower plasma membrane fluorescence intensity compared to MemGraft-Cy3; suspension cells show increased fluorescence intensity with increasing IFN-γ concentration, and stimulated NK-92 cells show higher fluorescence intensity than unstimulated cells or A549 cells.