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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 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.
HeLa cells were co-transduced with CellLight Early Endosomes-GFP and CellLight Late Endosomes-RFP and and incubated overnight.
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.
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 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.
Co-localization using LysoTracker Deep Red.
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.
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.
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. |
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 | Cy5 | Alexa Fluor 647 | 650/668 | 5 mg | D22914 |
Dextran, Alexa Fluor 680, 10,000 MW | Cy5.5 | 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
|
Cy5
|
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 |
No
|
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. |
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. |
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 | Cy5 | Alexa Fluor 647 | 650/668 | 5 mg | D22914 |
Dextran, Alexa Fluor 680, 10,000 MW | Cy5.5 | 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
|
Cy5
|
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 |
No
|
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. |