TRITC-DHPE

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
5-Nitro BAPTA, designed to a red fluorescent probe for cytoplasmic Ca²⁺ with strong emission in the long-wavelength region^[1].
TRITC-DHPE can be served as a unilamellar liposome, and labels cells^[1].

Protocol of flow cytometry assay^[1]:
1.Dissolve TRITC-DHPE probe in ethanol;
2.Sonicate and dilute the probe with electroporation buffer and sonicate the probe again, prepare the probe final stock concentration of 4.6 μM;
3.Add 15 mL probe stock solution into a T-75 flask, label cells at 37 °C for 2.5 h;
4.Wash cells twice with PBS, trypsinize cells, and wash cells again prior to analysis;
5.Pellet and resuspend cells in Ca²⁺-, Mg²⁺-free PBS, analyze via flow cytometry with 514 nm laser excitation and 585/42 nm filter;
6.Adjust aqueous suspension with pH range of 4-6.5;
7.Construct TRITC-DHPE pH-independent emission standard curves and set the excitation wavelength at 514 nm, set emission wavelength at 580 nm, and slit widths of 4 nm.

Protocol of TRITC-DHPE preparation^[2]:
1.Store TRITC-DHPE in chloroform (1 mg/ml, stock);
2.Dry dye stock solution (1-5 μL) into a film immediately before use, and reconstitute in 20–100 μL of ethanol;
3.Incubate cells with a final concentration of 100 nM-1 μM for 5-10 min at 22 °C in supplemented RPMI 1640 media with FCS;
4.The maximum concentration of ethanol during incubation was 1% v/v.

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