Endocytosis is a process by which cells internalize non-particulate materials such as proteins or polysaccharides by engulfing them. This process is important for metabolism and cell signaling. Fluorescent proteins and organic dyes are useful indicators for staining vesicle walls or vesicle contents. Because the vesicle pH changes during the endocytic process, pH indicators are useful to monitor stages in the pathway.

 

 


Fluorescent proteins

Fluorescent proteins can be used to specifically label components in the endocytic pathway such as early endosomes, late endosomes, or lysosomes. Using BacMam technology, the specific structures are targeted within the cell by transgenic expression of RFP or GFP fusion proteins. Endocytic structures can be monitored in live cells, or the cells can be fixed for additional immunohistochemical analysis.

BacMam technology is highly efficient, and transiently transduced cells typically express fusion protein for about five days, though in slowly dividing cells, such as some primary cell types, expression has been demonstrated for up to two weeks.

Endocytosis and pinocytosis

HeLa cells were co-transduced with CellLight® Early Endosmes-GFP and CellLight® Late Endosmes-RFP and and incubated overnight.


Dextran conjugates

Tracking internalization of fluorescent dextrans is a routine method for analyzing fluid-phase endocytosis. pHrodo™ dyes provide the most complete solution by allowing discrimination of stages in the endocytosis pathway from early endosome to lysosome formation with no quench or wash required.

pHrodo™ dyes are essentially non-fluorescent at neutral pH and exhibit increasing signal with a red or green readout respectively as the pH decreases. The increase in fluorescent signal can be used to monitor progression in the endocytic pathway.

Alexa Fluor® conjugates can be used to monitor internalization by using either quenching or washing steps.

Endocytosis and pinocytosis

Mouse monocyte/macrophage cells (MMM cells, ATCC) incubated with pHrodo™ Red dextran 10,000 MW in Live Cell Imaging Solution and counterstained with NucBlue® Live ReadyProbes™ Reagent .


LysoTracker® dyes

LysoTracker® probes specifically label acidic organelles with organic dyes and without using a transgenic protein system.  Their stable fluorescent signal covers a range of wavelengths but does not distinguish between lysosomes and other acidic compartments.

LysoTracker® probes can be multiplexed with other dyes or proteins. LysoTracker® Deep Red, in particular, multiplexes well with both RFP and GFP.

Both membrane proteins and LysoTracker® dyes serve as effective tools for tracking lysosome behavior. Use the selection guides to determine multiplexing options and fixability.

endocytosis-lysotracker

Co-localization using LysoTracker® Deep Red.


Membrane stains

An additional tool to track endocytosis uses membrane probes to monitor vesicle formation by visualizing changes in membrane morphology.

Water-soluble FM® dyes are lipophilic, nontoxic to cells and virtually nonfluorescent in aqueous medium. They insert into the outer leaflet of the cell membrane and become intensely fluorescent in a range of colors. FM® dyes will stain the plasma membrane and become internalized in endocytic vesicles. The non-specific staining of cell surface membranes can be washed off prior to imaging.

FM® 1-43 is particularly used to investigate the mechanisms of activity-dependent vesicle cycling especially in actively firing neurons.

Endocytosis and pinocytosis

Bovine pulmonary artery endothelial cells stained with the reactive oxygen species indicator, 6-carboxy-2´,7´-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) and FM® 5-95 and Hoechst 33342.

The red-fluorescent FM® 5-95 appears to stain both the plasma membrane and early endosomes; the green-fluorescent, oxidized carboxydichlorofluorescein localizes to the cytoplasm; and the blue-fluorescent Hoechst 33342 dye stains the nucleus.

Endocytosis and pinocytosis selection guides

 
Readout
Stable signal from fluorescent protein targeted to the specific stage of endocytosis
Range
Early endosome
Late endosome
Lysosome
Common filter set
FITC
TRITC
FITC
TRITC
FITC
TRITC
Labels
GFP
RFP
GFP
RFP
GFP
RFP
Ex/Em (nm)
488/510
555/584
488/510
555/584
488/510
555/584
Signal-to-noise ratio
Photostability
Bibliography
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Platforms
Imaging 
Format
1 mL
1 mL
1 mL
1 mL
1 mL
1 mL
Cat. No.
 
Alexa Fluor® dextrans
Readout
No-wash, no-quenching fluorescence intensity assay format where fluorescence increases throughout endocytosis 
Stable fluorescent signal irrespective of pH changes during endocytosis. Requires wash step or quenching of extra-cellular signal
Range
Monitors early endosome to early lysosome formation
No signal modulation with endocytic stages 
Vehicle
Dextran 10,000 MW
Dextran 10,000 MW
Dextran 10,000 MW
Common filter set
TRITC
FITC
Multiple Wavelength choice
Labels
pHrodo™ Red
pHrodo™ Green
Alexa Fluor® dye series
Ex/Em (nm)
560/585
509/533
Multiple
Signal-to-noise ratio
Photostability
Bibliography
Multiplexing
Yes
Yes
Live cells
Yes
Yes
Fixed cells
No
No
Fixable
Yes
Anionic fixable
Platforms*
I, M, FC
I, M, FC 
Format
0.5 mg
0.5 mg
Various
Cat. No.
* I = Imaging, M = Microplate, FC = Flow cytometry.

 

Alexa Fluor® dextrans selection guide

 
Standard filter set(s)
Fluorophore/conjugate
Ex/Em (nm)
Quantity
Cat. No.
Dextran, Alexa Fluor® 488, 10,000 MW FITC Alexa Fluor® 488 495/519 5 mg D22910
Dextran, Alexa Fluor® 546, 10,000 MW TRITC Alexa Fluor® 546 556/573 5 mg D22911
Dextran, Alexa Fluor® 555, 10,000 MW Alexa Fluor® 555 555/565 5 mg D34679
Dextran, Alexa Fluor® 568, 10,000 MW Rhodamine Alexa Fluor® 568 578/603 5mg D22912
Dextran, Alexa Fluor® 594, 10,000 MW Texas Red® Alexa Fluor® 594 590/617 5 mg D22913
Dextran, Alexa Fluor® 647, 10,000 MW Cy®5 Alexa Fluor® 647 650/668 5 mg D22914
Dextran, Alexa Fluor® 680, 10,000 MW Cy®5.5 Alexa Fluor® 680 679/702 5 mg D34680
Dextran, Tetramethylrhodamine and Biotin, 10,000 MW TAMRA TAMRA 555/580 10 mg D3312
Dextran, Fluorescein and Biotin, 10,000 MW FITC Fluorescein 494/518 10 mg D7178
 
Readout
Localization of lysosomes by fluorescence intensity measurement
Range
Targets acidic organelles at nanomolar concentrations
Common filter set
FITC
TRITC
Cy®5
Fluorescent label
LysoTracker® Green
LysoTracker® Red
LysoTracker® Deep Red
Ex/Em (nm)
504/511
577/590
647/668
Signal-to-noise ratio
Photostability
Bibliography
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Yes
No
Platforms*
I, M, FC
Format
20 x 50 μL
20 x 50 μL
5 x 50 μL
Cat. No.
* I = Imaging, M = Microplate, FC = Flow cytometry.
 
Readout
Image vesicle membranes by fluorescence intensity
Range
Requires quench or wash to remove non-specific signals from cell surface membranes
Common filter set
TRITC
TRITC
Texas Red®
Long pass 580
Long pass 580
Long pass 580
Labels
FM® 1-43
FM® 1-43FX
FM® 2-10
FM® 4-64
FM® 4-64FX
FM® 5-95
Ex/Em (nm)
471/581
471/581
506/620
558/734
565/744
560/734
Signal-to-noise ratio
Photostability
Bibliography
Multiplexing
No
Yes
No
No
Yes
No
Live cells
Yes
Fixed cells
No
Fixable
No
Yes
No
No
Yes
No
Platforms
Imaging
Format
10 × 100 µg
10 × 100 µg
5 mg
10 × 100 µg
10 × 100 µg
1 mg
Cat. No.