High Specificity for Lysosomes

The lysosomal localization of competitor's dye and pHLys Red was compared using HeLa cells expressing the lysosomal marker protein LAMP1-GFP. The competitor's dye stained HeLa cells showed higher background due to the leakage from lysosomes, whereas pHLys Red showed lower background (Merged image). The pHLys Red was found to be more accurately localized to lysosomes compared to the competitor's dye.

＜Experimental Conditions＞
Green: Ex= 488 nm, Em= 500-570 nm
Red : Ex= 561 nm, Em= 560-620 nm

High Retention in Lysosome

The lysosomal retention ability of pHLys Red and competitor's dye was compared using cells stained with each dye. The fluorescence of the competitor's dye decreased 24 hours after staining, whereas the fluorescence of pHLys Red was maintained and retention was high.

＜Experimental Conditions＞
Green: Ex= 488 nm, Em= 490-550 nm
Red: Ex= 561 nm, Em= 560-620 nm

Application Data: Lysosomal Mass and pH Exchange in Senescence-induced Cells

We analyzed senescence-associated acidic β-galactosidase (SA-βGal) activity and lysosomal mass and pH in A549 cells treated with Doxorubicin (DOX) to induce senescence. The SA-βGal activity was detected by SG03 Cellular Senescence Detection Kit - SPiDER-βGal, the lysosomal mass and pH were detected separately with L264 LysoPrime Deep Red and pHLys Red. Fluorescence imaging showed that the increase in lysosomal mass and pH acidification were observed in senescence-induced cells, and the normalized fluorescence intensity of lysosomal mass and pH by plate reader measurement showed the same result.

＜Experimental Conditions＞
SA-βGal(Green):
Ex = 488 nm, Em = 490 – 550 nm
Lysosomal pH (Red):
Ex = 561 nm, Em = 560 – 620 nm
Lysosomal mass (Deep Red):
Ex = 633 nm, Em = 640 – 700 nm

＜Experimental Conditions＞
SA-βGal: Ex = 525 – 535 nm, Em = 550 – 570 nm
Lysosomal pH: Ex = 555 – 565 nm, Em = 590 – 610 nm
Lysosomal mass: Ex = 645 – 655 nm, Em = 690 – 710 nm