Phen Green SK diacetate

Protocol for characterization of copper transport on the plasma membrane of CSB cells ^[1]:

10 mM Phen Green SK storage solution was prepared with DMSO, and the storage solution was stored away from light at -20℃ or -80℃ after subpackaging, and used up within 2 weeks.

Preparation of 80µM Phen Green SK working solution was prepared by preheating serum-free cell medium or PBS. Please adjust the concentration of the working liquid according to the actual situation.

1.Flow cytometry experiments

Intact protoplasts isolated from both 0.1 µM (control) and 100µM CuSO₄-grown cells were labeled with the copper-sensitive probe Phen Green SK diacetate (PGSK) to demonstrate copper sequestration.

 Vacuoles were isolated from PGSK-loaded protoplasts to see if the probe could enter this organelle.

Samples were analyzed in flow cytometer equipped with an argon-ion laser emitting a 488 nm beam at 15 mW.

Green fluorescence was collected through a 488 nm blocking filter, a 550 nm long-pass dichroic filter and a 525 nm band-pass filter. For each sample, 5,000 protoplasts or vacuoles were analyzed at low flow rate.

2.Copper transport by CSB protoplasts with Phen Green SK diacetate

Copper transport across the plasma membrane was determined with PGSK using spectrofluorometer with excitation and emission wavelengths set at 506 and 532 nm, respectively.

For transport experiments, protoplasts (10⁶) were incubated with 80µM PGSK for 30 min to equilibrate the dye.

The loaded protoplasts (100 µl) were added to the assay cuvette with 9 vols. of the mannitol-containing buffer, pH 5.6. After stabilization of the fluorescence signal, different amounts of CuCl₂ were added (0.1-3.5 mM, final concentrations).

To discriminate the unspecific fluorescence quenching and the quenching derived from copper uptake by protoplasts, the chelators EDTA and BCS were added to the assay medium. The effect of Cu²⁺ on the hydrolyzed external probe was tested after treatment of PGSK-loaded protoplasts with 0.5% Triton X-100.

To study which form of copper ion was taken up by protoplasts, ascorbic acid was added to the assay medium, reducing Cu²⁺ to Cu⁺.

To assess the effect of various cations on Cu²⁺ uptake, protoplasts were incubated for 5 min with the chloride forms of Ca²⁺ (10 mM), K⁺ (10 mM), Mn²⁺ (5 mM), Na⁺ (10 mM) and Zn²⁺ (10 mM) before the addition of CuCl₂.

To test how Cu²⁺ uptake is regulated by copper availability in the medium, cells grown for 6 d were incubated for 8 h with 100 µM CuSO₄ or with 20 µM of the chelator BCS.

Protocol for measure intracellular ferrous iron levels ^[2]:

1.Cells were treated as indicated before 10 μM Phen Green SK diacetate was added and incubated for 10 min.

2.The cells were washed twice with PBS to remove the excess PGSK.

3.The cells were trypsinized and resuspended in PBS plus 5% FBS.

4.A flow cytometer was used for detection of ferrous iron. Each group was triplicated.

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1].  Martins V, Hanana M, et,al. Copper transport and compartmentation in grape cells. Plant Cell Physiol. 2012 Nov;53(11):1866-80. doi: 10.1093/pcp/pcs125. Epub 2012 Sep 5. PMID: 22952251.

[2].  Wang Z, Yao M, et,al. Dexmedetomidine attenuates myocardial ischemia/reperfusion-induced ferroptosis via AMPK/GSK-3β/Nrf2 axis. Biomed Pharmacother. 2022 Oct;154:113572. doi: 10.1016/j.biopha.2022.113572. Epub 2022 Aug 18. PMID: 35988428.