|Labeling of Endocytic Vesicles Using
Fluorescent Probes for Fluid-Phase
Endocytosis occurs by the invagination of plasma
membrane to form an intracellular vesicle. Endocytic
vesicles, and the intracellular organelles they communicate
with, can be labeled by inclusion of fluorescent,
membrane-impermeant molecules in the extracellular
medium. Such probes are enclosed in the vesicles as
they form and remain contained in endocytic compartments.
Ultimately, these fluid-phase endocytic
probes either accumulate in lysosomes, where they
may be degraded, or are returned to the extracellular
medium by recycling vesicles. Fluid-phase endocytosis
is also called pinocytosis, which is sometimes subdivided
into macropinocytosis and micropinocytosis,
the formation of large and small pinosomes, respectively
(Maniak, 2001; Swanson, 1989a; Swanson and
Watts, 1995). Macropinocytosis is specialized for fluidphase
uptake, whereas micropinocytosis involves
both receptor-mediated (membrane adsorptive) and
fluid-phase uptake. Receptor-mediated endocytosis
via clathrin-coated vesicles can be distinguished from
fluid-phase endocytosis by using fluorescent specific
ligands for the receptor, e.g., FITC-labeled transferrin
for the transferrin receptor. Compared with receptormediated
endocytosis, the biological function of
macropinocytosis has been less investigated so far.
However, attention has been increasingly paid to this
fluid-phase endocytic pathway (Araki et al.
, 2000, 2003;
Maniak, 2001) because it has been known that
macropinocytosis is utilized for antigen presentation
in dendritic cells and macrophages (Norbury et al.
1995; Sallusto et al.
, 1995; Nobes and Marsh, 2000).
A variety of fluorescent probes can be used to label
endocytic organelles in living cells. Vital staining
techniques using such probes are useful not only for
marking endocytic organelles, but also for exploring
the kinetics of soluble molecules and the dynamics of
endocytic organelles in the cell. This article describes
methods for labeling and observing these compartments
in living cells and for immunofluorescence
microscopy of similarly labeled cells. In addition, it
describes a method for the quantitative measurement
of fluid-phase pinocytosis using fluorescent probes.
These methods are optimized for the study of
macrophages, which are actively endocytic cells. It
should be kept in mind that most other kinds of cells
exhibit lower rates of endocytosis and different kinetics
of delivery from endosomes to lysosomes. One may
therefore have to extend incubation times or increase
probe concentrations to obtain strong fluorescent
signals in the microscope or fluorometer. For any new
cell type, compartment identities should be determined
empirically by comparing fluorescent probe
distributions in cells fixed after various pulse-chase
intervals with the immunofluorescent localization of
known markers for endocytic compartments (e.g.,
Racoosin and Swanson, 1993).
II. MATERIALS AND
The fluorescent fluid-phase probes, fluorescein
dextran, MW 3000 (FDx3, Cat. No. D-3305), MW 10,000
(FDxl0, Cat. No. D-1821), lysine fixable fluorescein dextran, MW 10,000 (Cat. No. D-1820), and Texas red
dextran, MW 10,000 (TRDxl0, Cat. No. D-1828), are
from Molecular Probes. Lucifer yellow CH (Cat. No.
86150-2) is from Aldrich. Fluorescein dextran, MW
150,000 (FDx150 Cat. No. FD-150), paraformaldehyde
(Cat. No. P-6148), bovine serum albumin (BSA, fraction
V., Cat. No. A-9647), Triton X-100 (Cat. No. T-
9284), saponin (Cat. No. S-4521), HEPES (Cat. No.
H-3375), and Trizma base (Cat. No. T-1503) are from
Sigma Chemical. The 25% glutaraldehyde solution
(EM grade, Cat. No. SP-17003-92) is from Nacalai
Tesque. A primary antibody, rabbit anti-cathepsin D
serum, was a gift from Dr. S. Yokota, Yamanashi
Medical School. Monoclonal antibodies recognizing
some marker proteins for endocytic compartments are
available from the Developmental Studies Hybridoma
Bank or commercial sources. Texas red-labeled antirabbit
IgG (goat) (Cat. No. TI-1000) is from Vector Lab.
Dulbecco's modified essential medium (DMEM, Cat.
No. 31600-034), fetal bovine serum (FBS, Cat. No.
16000), and goat serum (Cat. No. 16210) are from
GIBCO BRL. Circular glass coverslips 12 and 25 mm in
diameter (No. 1 thickness, Cat. No. 12-545-102) and
silicon oil (Cat. No. 5159-500) are from Fisher Scientific.
Twenty-four-well (Cat. No. 430262) and 6-well (Cat.
No. 430343) plates are from Corning Costar. A water
aspirator with a Pasteur pipette and a digital
micropipette (Nichipet EX1000) are used for quickly
replacing media in cell culture wells. The Attofluor cell
chamber (Cat. No. A7816) is from Molecular Probes.
PermaFluor aqueous mounting medium (Cat. No.
434980) is from Lipshaw/Immunon.
An epifluorescence microscope (Axiophot, Carl
Zeiss) is used for observation of both living and fixed
cells. A Lucifer yellow filter set is from Omega Optical
(Set No. XF-14), in addition to a conventional fluorescein
filter set. For high-resolution observation, a 100× PlanApo lens, numerical aperture (NA) 1.4 or a 100× Plan-Neofluar lens, NA 1.3 is used. An inverted-type
fluorescence microscope (Nikon TE300) equipped with
a cooled CCD camera (Retiga EXi F-M-12-C, QImaging),
light path shutter (Uniblitz VMMD1, Vincent
Associates), and a thermo-controlled stage (Model TC-
102, Harvard Apparatus) are used for longer observations
of living cells. A personal computer (Precision
360, Dell) installed with MetaMorph version 4.6
imaging system software (Universal Imaging Co.) is
used for collecting images from the CCD camera and
for making time-lapse movies. For quantitation of fluorescent
probes, a spectrofluorometer (Hitachi 650-40)
A. Fluorescence Microscopy of Endocytic
Compartments Labeled with Fluorescent
- Ringer's buffer + bovine serum albumin (BSA) (RB):
155mM NaCl, 5mM KCl, 2mM CaCl2, 1 mM MgCl2,
2 mM NaH2PO4, 10 mM HEPES, 10 mM D-glucose,
pH 7.2, plus 0.05% BSA. To make 1 liter, add 9.1 g of
NaCl, 0.37g of KCl, 0.275g of NaH2PO4·H2O, 2.38g
of HEPES, 1.8g of D-glucose, 0.22 g of CaCl2, 0.2 g of
MgCl2·6H2O, and 0.5g BSA to 950ml distilled water,
adjust to pH 7.2 with 1 N NaOH, and bring the volume
to 1 liter. Sterilize with a 0.22-µm filter and store at 4°C.
- Fluorescent probe stock solutions: Dissolve 10mg
of fluorescent probes such as Lucifer yellow, FDx3,
FDx10, FDx150, and TRDxl0 in 1 ml PBS. Lysine
fixable dextran probes can be used to increase fluorescent
signals in aldehyde-fixed cells. Divide in 200-µl
aliquots in tubes and store at -20°C.
- Labeling medium: Dilute fluorescent probe solutions
to a final concentration at 1.0mg/ml in RB. A
volume of 0.4 ml labeling medium is required for each
well of a 24-well dish. The concentrations of probes
may be changed depending on cell types. One can differentially
label macropinosomes and micropinosomes
using different sized probes (Fig. 1). To label both
macropinosomes and micropinosomes, use low molecular
weight probes such as Lucifer yellow and FDx3.
To label primarily macropinosomes, use larger probes,
such as FDx150 (Araki et al., 1996). Warm to 37°C before adding to cells.
- 8% paraformaldehyde stock solution: To make 50 ml,
add 4 g paraformaldehyde to 30ml distilled water and
heat to 70°C while stirring. Add a few drops of 1N NaOH so that the mixture becomes clear. Bring the
final volume to 50ml with distilled water and filtrate
with paper filter. This solution may be kept in aliquots
at -20°C. To make up a fixative, thaw an aliquot in a
hot water bath (~50°C) until the solution becomes clear.
- 80mM HEPES stock solution, pH 7.2: To make
100ml, add 1.91g of HEPES to 70ml of distilled
water. Adjust pH to 7.2 with 1 N NaOH while stirring.
Bring the final volume to 100ml with distilled water.
Store at 4°C.
- Fixative: 4% paraformaldehyde and 0.1% glutaraldehyde
in 40mM HEPES buffer, pH 7.2, containing
6.8% sucrose. To make 10ml, add 5ml of 8%
paraformaldehyde solution, 40µl of 25% glutaraldehyde
solution, and 0.68g of sucrose to 5 ml of 80mM HEPES, pH 7.2.
- Phosphate-buffered saline (PBS): To make 5 liters,
dissolve 40 g of NaCl, 1 g of KCl, 7.1 g of Na2HPO4, and
1 g of KH2PO4 in 4 liters of distilled water. Bring the
final volume to 5 liters with distilled water. The solution
should be pH 7.2.
- Culture cells on 12-mm, circular, No. 1 thickness
coverslips in 24-well culture dishes in DMEM with
10% heat-inactivated FBS.
- Aspirate the culture medium from the cell
culture well and add prewarmed labeling medium.
Swirl the dishes and incubate cells in labeling medium
for various times at 37°C. Then rinse quickly by changing
warm RB to remove fluorescent probe and chase
in RB as necessary. For mouse macrophages, a 2- to 5-
min labeling incubation without chase will label early
endosomes, including micro- and macropinosomes
(Fig. 1). A subsequent chase in the absence of the probe
for 5 to 15 min should label late endosomes. A 30- to
60-min labeling incubation labels all endocytic compartments,
including early and late endosomes and
lysosomes, and a 30-min labeling followed by a chase
longer than 60min should label primarily lysosomes
- Fix the cells in 4% paraformaldehyde and 0.1%
glutaraldehyde in 40mM HEPES, pH 7.2, containing
6.8% sucrose for 30 to 60min at 37°C.
- Rinse 3 × 5 min with PBS.
- Mount the coverslip, cell side down, on a slide
using mounting medium. Seal with nail polish
between the coverslip and the slide.
- Observe the slide with an epifluorescence microscope.
We can observe living cells without fixation for
short periods using a simple microscope culture
chamber. This method has been described in detail
previously (Swanson, 1989b; Raccosin and Swanson,
1994). Briefly, assemble a chamber on a slide using
small coverslip fragments to support the coverslip.
RB should be added to fill the space between the slide
and the coverslip. Seal the coverslip to the slide using
a heat-melted paraffin-based compound (Swanson,
1989b). The method for longer observations of living
cells is described next.
|FIGURE 1 Differential labeling of macropinosomes and micropinosomes with Lucifer yellow (A) and
FDx150 (B). Macrophages were incubated for 5 min in labeling medium containing Lucifer yellow or FDx150,
washed briefly, and fixed immediately. A low molecular weight probe, Lucifer yellow (MW 457), labels both
macropinosomes and micropinosomes (A). A larger probe, FDx150 (MW 150,000), labels predominantly
(B). Bars: 10µm.
|FIGURE 2 Immunofluorescence of cathepsin D in endocytic compartments labeled with endocytic probes.
Macrophages were incubated for 30min in labeling medium containing 0.5mg/ml lysine-fixable FDxl0 and
were chased for 30min. Cells were then fixed and processed for immunofluorescence using a primary antibody
against cathepsin D and the Texas red-conjugated secondary antibody. (A) Fluorescein image shows
that FDxl0 labels tubular lysosomes. (B) Texas red image shows that tubular lysosomes, which are labeled
with FDxl0, are positive for cathepsin D. Bar: 10µm.
B. Observation of Endocytic Compartments
in Living Cells
C. lmmunocytochemical Characterization of
Endocytic Compartments Labeled with
a Fluorescent Probe
- Plate cells on 25-mm No. 1 coverslips in a 6-well
culture dish containing tissue culture medium.
- Replace the culture medium with labeling
medium. Incubate in labeling medium for various
times at 37°C as described earlier, to label the endocytic
- Wash away fluorescent probe from the coverslip
- Assemble the coverslip in an Attofluor chamber
and fill with 1 ml of RB. You may slowly add a small
amount of silicon oil to cover the surface of RB in the
chamber. The thin layer of silicon oil protects evaporation
of RB during long observations.
- Put one drop of immersion oil on the objective
lens of an inverted microscope and settle the Attofluor
chamber in the Leiden chamber thermocontrolled at
37°C on the microscope stage.
- Observe the coverslip and acquire images using
a high sensitive cooled CCD camera under the lowest
light exposure as possible, as intense excitation light
may cause not only photobleaching, but also photochemical
damage to living cells. Under optimal conditions
of labeling, we can observe cells for several
minutes under conditions of low-intensity illumination.
This can be extended to an hour by inserting a
shutter into the light path to control exposures.
- Collect time-lapse images using MetaMorph
imaging software, which controls the CCD camera and
the light path shutter. These collected images can be
saved as a stack file in electronic storage media such
as CD-R or DVD-RAM for later processing into movies
of living cells. For further information of imaging techniques, see also the sections on digital video
microscopy and fluorescent microscopy of living cells
in Volume 3.
- Fixative: Prepare 10ml of 4% paraformaldehyde in
40mM HEPES buffer, pH 7.2, containing 6.8%
- 0.25% NH4Cl in PBS (NH4Cl/PBS): To make 100ml,
dissolve 0.25 g of NH4Cl in 100 ml PBS
- 0.25% saponin or Triton X-100, 2% BSA in PBS (permeabilizing/
blocking buffer): To make 50ml, dissolve
1 g BSA and 125 mg saponin or Triton X-100 in 50 ml
- Primary antibody: Dilute serum or antibody with
permeabilizing/blocking buffer. In the example
shown, we diluted rabbit anti-cathepsin D serum at
- Secondary antibody: Dilute Texas red-labeled antirabbit
IgG with PBS at 1:250-500
D. Quantitative Fluorometric Analysis of
Endocytic Compartments Labeled with
- Incubate the cells with labeling medium as
described earlier. Lysine-fixable FDx or Lucifer yellow
should be used when the other markers are to be localized
by immunofluorescence. Nonfixable FDx would
be lost during permeabilization of the cell.
- Rinse in PBS to remove excess fluorescent probe,
unless the cells were chased in RB without fluorescent
- Fix in 4% paraformaldehyde in 40mM HEPES
buffer, pH 7.2, containing 6.8% sucrose for 30-60min
at 37°C. Glutaraldehyde may be added to the fixative
(0.1%), providing that antigenicity is resistant to
- Rinse with PBS 3 × 5 min and further immerse in
NH4Cl/PBS for 10min to quench free aldehyde.
- Treat cells with permeabilizing/blocking buffer
for 2 × 5 min.
- Put parafilm in a container with a moist paper.
Place one 40-µl drop of primary antibody on the
parafilm for each coverslip.
- Wipe the cell-free side of the coverslip with
Kimwipe paper. Place the coverslip cell side down on
a drop of primary antibody. Incubate with the primary
antibody for 1 h at room temperature in the moisture
chamber. You may prolong the incubation time to
overnight at 4°C.
- After incubation, put the coverslip back into the
well and wash three times for 5 min each with PBS.
- Using the same method as for the primary antibody,
incubate with secondary antibody, e.g., Texas
red-conjugated anti-rabbit IgG diluted in PBS at a concentration
1:500, for 1 h at room temperature.
- Wash the coverslip with PBS three times for
5 min each.
- Mount the coverslip on a slide using the mounting
medium. Seal the coverslip with nail polish.
Observe the specimens with an epifluorescence microscope
using fluorescein and Texas red filter sets.
- Lysis buffer: 0.1% Triton X-100 in 50mM Tris, pH
8.5. To make 100ml, dissolve 0.6 g of Trizma base and
0.1 g of Triton X-100 in 80 ml of distilled water. Adjust
pH to 8.5 with 1 N NaOH. Bring the volume to 100ml.
- PBS: Make 3 liters of PBS as described earlier.
Refrigerate before use.
- 0.1% BSA/PBS: To make 2 liters, dissolve 2g of
BSA in 2 liters of PBS.
- Standard solutions of fluorescent probes: Dilute the
labeling medium to concentrations of 0, 1, 5, 10, and
20ng probe/ml in lysis buffer. Each solution should be
more than 2 ml.
- Plate the cells at a high density (e.g., 2 × 105 cells/well) in a 24-well culture dish. Triplicate experiments
- Replace the culture medium with labeling
medium containing fluorescent probes. Dual labeling
with FDx and Lucifer yellow is possible (Berthiaume et al., 1995). Incubate at 37°C for various times. A 0-min
incubation should be done as a control to determine
the background level.
- Discard the labeling medium and rinse the
culture dish twice by dipping into a l-liter beaker filled
with ice-cold 0.1% BSA/PBS for 5min each. Repeat
with another beaker filled with cold PBS for 5 min.
- Drain PBS and aspirate remaining PBS
- Put 0.5 ml of lysis buffer into each well and leave
it at least 30min to complete cell lysis.
- Fill disposable 1-cm plastic cuvettes with 0.75 ml
of lysis buffer. Add 0.4 ml of cell lysate into the plastic
cuvette and dilute it with another 0.75 ml of lysis buffer
so that the final volume is 1.9ml.
- Measure the fluorescence of lysate in a spectrofluorometer.
Fluorescein can be measured at excitation
(exc.) 495nm and emission (em.) 514nm. Lucifer
yellow is exc. 430nm and em. 580nm. These wavelengths
allow selective measurement of FDx and
Lucifer yellow when the cells are labeled with both
probes. Lucifer yellow alone is best measured at exc.
430 nm and em. 540 nm.
- Measure the protein concentration of lysates
remaining in wells using a BCA protein assay kit
(Pierce Chemical Co.).
- Prepare the standard solutions of 0, 1, 5, 10, and
20ng probe/ml in lysis buffer and measure in a spectrofluorometer
to obtain a standard curve.
- Calculate the amount of probes from the standard
curve and express the value as nanogram probe
per milligram protein.
- A prolonged exposure to intense excitation light
may cause a release of fluorescent probes from endocytic
vesicles into cytoplasm, especially in living cells.
To avoid this, reduce the intensity of the excitation
light or the exposure time as much as possible (Video
1 and 2 in the online version).
- Lucifer yellow can be seen using some fluorescein
filter sets with a wide band pass (e.g., Olympus
BP490), but not some others (e.g., Zeiss No. 09).
Choose an appropriate filter set for Lucifer yellow
(e.g., Omega Optical Set No. XF-14, Zeiss No. 05).
- Many kinds of fluorescent-conjugated probes
are available commercially; however, some are not
suitable for fluid-phase probes. Texas red albumin is
taken up very efficiently by adsorptive endocytosis,
although it is sometimes used as a fluid-phase probe.
Lysine-fixable Texas red dextran may bind nonspecifically
to coverslips and create a high background
- In the combination of fluorescent fluid-phase
probe labeling with immunocytochemistry, we are
often faced with a problem that fluid-phase probes
release from macropinosomes during membrane permeabilization
that is necessary for antibody impregnation.
In such a case, the author recommends using a
mild detergent such as saponin rather than Triton X-
100. The addition of 2% BSA to permeabilizing buffer
considerably protects the fluid-phase probe release.
The addition of 0.1% glutaraldehyde to the fixative
also effectively fixes fluorescent probes in macropinosomes;
however, because some antigens lose their antigenicity, you have to examine the resistance of the
antigen to glutaraldehyde.
- For quantitative analysis using a less sensitive
fluorescence plate reader, plate the cells at a higher
density to increase the sensitivity for fluorescence.
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