I am doing an experiment to monitor the pH of lysosomes in cells and want to try the probe CypHer 5.
But there are some questions:
First, is it a live cell dye?
After all, we want to track the dynamic changes of lysosomes in living cells, and it would be inconvenient if it can only dye fixed cells; the other question is whether it can pass through the plasma membrane and enter the cell by itself?
If it needs to use transfection reagents or other treatments, then the experimental steps have to be adjusted.
Binbin Lee
Yes, CypHer 5 is a pH-sensitive fluorescent probe suitable for living cells.
It does not require fixation or permeabilization and can monitor receptor internalization in real time in living cells. However, CypHer 5 requires receptor agonists to induce GPCRs in living cells to internalize CypHer 5. The fluorescence signal of CypHer 5 depends on the environmental pH: in neutral pH (such as the extracellular environment or cell membrane surface), the probe hardly emits light (non-fluorescent state); in acidic environments (such as endosomes, lysosomes, pH≈6.1 and below), the probe is protonated and emits strong fluorescence (PMID: 15090190).
Principle (PMID: 15090190):
When the CypHer 5-labeled antibody binds to the target receptor on the cell surface, in the unactivated state, the fluorescence signal is extremely low due to the neutral extracellular environment; when the receptor is activated by an agonist and enters the acidic endosome or lysosome through endocytosis, CypHer 5 emits fluorescence due to the acidification of the environment, thereby reflecting the internalization and trafficking pathway of the receptor through changes in fluorescence intensity.
Method (PMID: 15090190):
Cell pretreatment
Live cells expressing the target receptor (such as CHO-K1 cells stably transfected with TRHR-1 or HEK 293 cells transfected with β₂-adrenergic receptor) are seeded in 96-well plates and cultured to 70-80% confluence.
Wash the cells twice with Krebs-Ringer buffer (pH 7.4) to remove serum interference.
Probe incubation and agonist treatment
Add CypHer 5 labeled antibody (concentration of about 5-10 μg/mL) and incubate at room temperature for 10-15 minutes to allow the antibody to bind to the cell surface receptor.
Add receptor agonist (such as TRH or isoproterenol) and incubate at 37°C for 30 minutes to induce receptor internalization.
The answer to this question comes from MedChemExpress (MCE) Technical Support.