Schematic of cell structure with expanded view of an endosome

Endosome structures are intracellular sorting organelles involved in trafficking proteins within the cell. Here we describe fluorescent fusion proteins that can be used to identify early and late endosomes in live cells.

See endosome markers selection guide

Endosome introduction

Endosomes are intracellular sorting organelles that transport subcellular components such as proteins and lipids from the Golgi [1]. Proteins can also be internalized through the plasma membrane in a process known as endocytosis, pinocytosis, and phagocytosis [1]. Learn more about these processes here. Endosomes are classified as early or late based on their stage following the internalization of cellular components. They are also distinguished by their morphology and Rab marker. Early endosomes are commonly formed near the plasma membrane and are distinguished by the Rab5A marker. Late endosomes mature from early endosomes and are distinguished by the Rab7A marker.

Selection guide for endosome stains

 Early endosome stainsLate endosome stains
Name
Readout
Stable signal from fluorescent protein targeted to the specific stage of endocytosis
Range
Early endosome
Late endosome
Common filter set
FITC
TRITC
FITC
TRITC
Labels
GFP
RFP
GFP
RFP
Ex/Em (nm)
488/510
555/584
488/510
555/584
Signal-to-noise ratio
Photostability
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Format
1 mL
1 mL
1 mL
1 mL
Cat. No.

Early and late endosome fluorescent proteins

CellLight fluorescent fusion proteins are ready-to-use constructs that label endosomes in live cells to follow the dynamics of intracellular behavior. CellLight Early Endosome labels use the Rab5a construct fused to emGFP (Figure 1) or TagRFP (Figure 2) while CellLight Late Endosome labels use the Rab7a construct fused to emGFP (Figure 3) or TagRFP (Figure 4).

Introducing CellLight fluorescent fusion proteins involves a simple transfection step using the BacMam technology, and they work like cell stains with minimal toxicity or chemical disruption. These endosome fusion proteins are compatible with other fluorescent probes for multiplex analysis in live cells, or after formaldehyde fixation for colocalization studies.

Early endosome images

Microscopic image of cell stained with green endosomes, red peroxisomes, blue lysosomes, and magenta nucleus
Figure 1. Early endosome-GFP co-localization with LysoTracker Deep Red. HeLa cell expressing CellLight Peroxisome-RFP and CellLight Early Endosome-GFP was loaded with 50nM LysoTracker Deep Red and Hoechst 33342. Images were pseudo colored as Hoechst 33342 (Magenta), LysoTracker Deep Red (Blue), CellLight Early Endosome-GFP (green) and CellLight Peroxisome-RFP (Red).
Microscopic image of cells stained with red endosomes, green mitochondria, and blue nuclei

Figure 2. Early Endosomes-RFP and mitochondria-GFP. HeLa cells were transduced with CellLight Endosomes-RFP and Mitochondria-GFP, then and co-stained Hoechst 33342. Imaging was performed on live cells using a DeltaVision Core microscope and standard DAPI/FITC/TRITC filter sets.

Late endosome images

Microscopic image of cells stained with green endosomes, red Golgi, and magenta mitochondria

Figure 3. Late endosome-GFP and Golgi-RFP. A549 cells were co-transduced with CellLight Late Endosomes-GFP and CellLight Golgi-RFP then incubated overnight. Following staining with MitoTracker Deep Red, cells were imaged on a Zeiss LSM confocal microscope.

Microscopic image of cells stained with red endosomes, green tubulin, blue lysosomes, and magenta nuclei

Figure 4. Late endosome-RFP co-localization with LysoTracker Deep Red. HeLa cells expressing CellLight Tubulin-GFP and CellLight Late Endsome-RFP was loaded with 50 nM LysoTracker Deep Red and Hoechst 33342. Images were pseudo colored as Hoechst 33342 (magenta), LysoTracker Deep Red (blue), CellLight Tubulin-GFP (green), and CellLight Late Endsome-RFP (red).

Learn more about endosome stains

References:

  1. Otegui M and Reyes FC (2010). Nature Education. 3(9):23.

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