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 (Swanson, 1989a; Swanson and Watts, 1995). A variety of fluorescent probes can be used to label endocytic organelles in living cells. We describe here methods for labeling and observing these compartments in living cells and for immunofluorescence microscopy of similarly labeled cells. In addition, we describe a method for 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 born 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, we recommend that compartment identities 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 (eg, Racoosin and Swanson, 1993). Monoclonal antibodies recognizing some of these marker proteins are available from the Developmental Studies Hybridoma Bank.
The fluorescent fluid-phase probes, fluorescein dextran, MW 3,000 (FDx3, Cat.No. D-3305), MW 10,000 (FDx10, Cat. No. D-1821), and lysine fixable fluorescein dextran, MW 10,000 (Cat.No. D-1820) were purchased from Molecular Probes. Lucifer yellow CH (Cat. No. 86150-2) was 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), HEPES (Cat. No. H-3375) Trizma base (Cat. No. T-1503) and p-phenylenediamine (Cat.No. P-6001) were from Sigma. A primary antibody, rabbit anti-cathepsin D serum, was a gift from Dr. S. Yokota, Yamanashi Medical School. Texas red-labeled anti-rabbit IgG (goat) (Cat. No. TI-1000) was from Vector Lab. Dulbecco's modified essential medium (DMEM, Cat. No. 31600-034), fetal bovine serum (FBS, Cat.16000) and goat serum (Cat.No. 16210) were from GIBCO BRL. Circular glass cover slips of 12- and 25 mm in diameter (No. 1 thickness, Cat. No. 12-545-102), silicon oil (Cat. No. 5159-500) were from Fisher Scientific. 24-well (Cat. No.430262) and 6-well (Cat.No.430343) plates were from Corning Costar. An epifluorescence microscope (Axiophoto, Carl Zeiss) was used for observation both of living and fixed cells. Lucifer yellow filter set was obtained from Omega Optical (Set No. XF-14), in addition to usual fluorescein filter set. For high resolution observation, a 100X Planapo lens, numerical aperture (NA) 1.4 or a 100X Plan-Neofluar lens, NA 1.3 was used. An inverted-type fluorescence microscope (Carl Zeiss IM-35) equipped with nuvicon video camera (ITC-510, Ikegami) and a multichannel plate intensifier (Model KS-1380, Video Scope Int'l) and thermo-controlled stage (Model TC-102, Medical Systems Corp.) were used for longer observations of living cells. A personal computer installed with image analysis software (MetaMorph 2.0, Universal Imaging Co.) was used for collecting images from the intensified video signal and for making time lapse movies.
III. Procedures
A. FLUORESCENCE MICROSCOPY OF ENDOCYTIC COMPARTMENTS LABELED WITH FLUORESCENT PROBES
Solutions
1. Ringer's buffer + BSA (RB): 155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 2 mM NaH2PO4, 10 mM HEPES, 10 mM D-glucose, pH 7.2 plus 0.05% BSA. To make 1 l, add 9.1 g of NaCl, 0.37 g KCl, 0.275 g of NaH2PO4H2O, 2.38 g of HEPES, 1.8 g of D-glucose, 0.22 g of CaCl2, 0.2 g of MgCl2á6H2O, and 0.5 g BSA to 950 ml distilled water, adjust to pH 7.2 with 1 N NaOH, and bring the volume to 1 l. Sterilize with 0.22 um filter and store at 4°C.
2. Labeling medium: To make 1 ml, dissolve 0.5 mg of lucifer yellow, FDx3, FDx10, or FDx150 in 1ml RB. 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, like FDx150 (Araki et al., 1996). Warm to 37C before adding to cells.
3. 8% paraformaldehyde stock solution: To make 50 ml, add 4 g paraformaldehyde to 30 ml distilled water and heat to 70C while stirring. Add a few drops of 1 N NaOH so that the mixture becomes clear. Bring the final volume to 50 ml with distilled water, and filtrate with paper filter. This solution may be kept in aliquots at -20C.
4. 80 mM HEPES stock solution, pH 7.2: To make 100 ml, add 1.91 g of HEPES to 70 ml of distilled water. Adjust pH to 7.2 with 1N NaOH while stirring. Bring the final volume to 100 ml with distilled water. Store at 4 C.
5. Fixative : 4% paraformaldehyde in 40 mM HEPES buffer, pH 7.2, containing 6.8% sucrose. To make 10 ml, add 5 ml of 8% paraformaldehyde solution and 0.68 g of sucrose to 5 ml of 80 mM HEPES, pH 7.2. Warm to 37C before applying to cells. 6. Phosphate buffered saline (PBS): To make 5 l, dissolve 40 g of NaCl, 1 g of KCl, 7.1 g of Na2HPO4, 1 g of KH2PO4 in 4 l of distilled water. Bring the final volume to 5 l with distilled water.
6. Mounting medium: To make 10 ml, put 10 mg p-phenylenediamine, 1ml of PBS, and 9 ml of glycerol in a 15-ml tube. Wrap the tube with aluminum foil to protect from light, and mix overnight on rotating wheel. Divide into aliquot tubes and store at -20C.
Steps
1. Culture cells on 12 mm, circular, No.1 thickness cover slips in 24 well culture dishes in DMEM with 10% heat-inactivated FBS.
2. Replace the culture medium with pre-warmed labeling medium. Swirl the dishes and incubate cells in labeling medium for various times at 37°C. Then quickly rinse 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 - 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 30 min should label only lysosomes (Fig. 2).
3. Fix the cells in 4% paraformaldehyde in 40 mM HEPES, pH 7.2, containing 6.8% sucrose for 30 to 60 min at 37C.
4. Rinse 3 x 5 min with PBS.
5. Mount the cover slip, cell-side down on a slide using mounting medium. Seal with a nail polish between the cover slip and the slide.
6. 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 previously described in detail (Swanson, 1989b; Raccosin and Swanson, 1994). Briefly, assemble a chamber on a slide using small cover slip fragments to support cover slip. RB should be added to fill the space between the slide and cover slip. Seal the cover slip to the slide using heat-melted paraffin-based compound (Swanson, 1989b). The method for longer observations of living cells is described below.
B. OBSERVATION OF ENDOCYTIC COMPARTMENTS IN LIVING CELLS
Step
1. Plate cells on 25 mm No.1 cover slips in a 6-well culture dish containing tissue culture medium.
2. Replace the culture medium with labeling medium. Incubate in labeling medium for various times at 37C, as described above, to label the endocytic compartments.
3. Wash away fluorescent probe from the cover slip with RB.
4. Assemble the cover slip in a Leiden chamber (Medical System Corp.), fill with 1 ml of RB and slowly add a small amount of silicon oil to cover the surface of RB in the chamber.
5. Put the chamber on the stage thermo-controlled at 37C.
6. Observe the cover slip with an inverted fluorescence microscope using the lowest light exposure as possible, since 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 up to an hour under conditions low-intensity illumination. This can be extended by inserting a shutter into the light path to control exposures.
7. Collect time-lapse images using a silicon-intensified target (SIT) or other sensitive video camera and MetaMorph 2.0 software (Universal Imaging Co.) on a personal computer. These images may be recorded on laser optical disks (Panasonic LQ-D5500) or electronic storage media for later processing into movies of living cells.
C. IMMUNOFLUORESCENCE IDENTIFICATION OF ENDOCYTIC COMPARTMENTS LABELED WITH A FLUORESCENT PROBE
Solutions
1. 0.25% NH4Cl in PBS (NH4Cl/PBS): To make 100 ml, dissolve 0.25 g of NH4Cl in 100 ml PBS.
2. 0.25% Triton X-100 in PBS (Tx/PBS): To make 300 ml, add 0.75 g of Triton X-100 to 300 ml PBS while stirring.
3. 2% heat-inactivated goat serum in Tx/PBS (HIGS-Tx/PBS): To make 10 ml, add 0.2 ml of heat-inactivated goat serum to 9.8 ml of Tx/PBS.
4. Primary antibody: Dilute serum or antibody with HIGS-Tx/PBS. In the example shown, we diluted rabbit anti-cathepsin D serum at 1: 500.
5. Secondary antibody: Dilute Texas red-labeled anti-rabbit IgG with HIGS-Tx/PBS at 1: 250 - 500.
Steps
1. Incubate the cells with labeling medium as described above. Lysine-fixable FDx or lucifer yellow should be used when the other makers are to be localized by immunofluorescence. Non-fixable FDx would be lost during permeabilizing of cells.
2. Rinse in PBS to remove excess fluorescent probe, unless the cells were chased in RB without fluorescent probe.
3. Fix in 4% paraformaldehyde in 40 mM HEPES, pH 7.2, containing 6.8% sucrose for 1 hr at 37C.
4. Rinse with PBS 3 x 5 min and further immerse in NH4Cl/PBS for 10 min to quench free aldehyde.
5. Treat cells with HIGS-Tx/PBS for 2 x 5 min for cell permeabilization and blocking.
6. Put parafilm in a container with a moist paper. Place one 40 ul drop of primary antibody on the parafilm for each cover slip.
7. Wipe the cell-free side of the cover slip with Kimwipe. Place the cover slip cell-side down on a drop of primary antibody. Incubate with the primary antibody for 1 hr at room temperature in the moisture chamber.
8. After incubation, put the cover slip back into the well and wash 3 times for 5 min each with Tx/PBS.
9. Using the same method as for primary antibody, incubate with secondary antibody, eg. Texas-red conjugated anti-rabbit IgG diluted in HIGS-Tx/PBS at a concentration 1: 500, for 1hr at room temperature.
10. Wash the cover slip with Tx/PBS 2 times and PBS once for 5 min each.
11. Mount the cover slip on a slide using the mounting medium. Seal the coverslip with nail polish. Observe the specimens with an epifluorescence microscope using fluorescein and rhodamine filter sets.
D. QUANTITATIVE FLUOROMETRIC ANALYSIS OF ENDOCYTIC COMPARTMENTS LABELED WITH FLUORESCENT PROBES
Solution
1. Lysis buffer: 0.1% Triton X-100 in 50 mM Tris, pH 8.5. To make 100 ml, dissolve 0.6 g of Trizma base, 0.1 g of Triton X-100 in 80 ml of distilled water. Adjust pH to 8.5 with 1N NaOH. Bring the volume to 100 ml.
2. 0.1%BSA/PBS: To make 2 l, dissolve 2 g of BSA in 2 l of PBS.
3. Standard solutions of fluorescent probes: Dilute the labeling medium to concentrations, 0, 1, 5, 10, 20 ng probe/ml in lysis buffer. Each solution should be more than 2 ml.
Steps
1. Plate the cells at a high density (eg. 2 x 105 cells/well) in a 24-well culture dish. Triplicate experiments are desirable.
2. 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‚b for various times. A 0-min incubation should be done as a control, to determine the background level.
3. Discard the labeling medium and rinse the culture dish twice by dipping into 1 l beaker filled with ice-cold 0.1% BSA/PBS for 5 min each. Repeat with another beaker filled with cold PBS for 5 min.
4. Drain PBS and aspirate remaining PBS completely.
5. Put 0.5 ml of lysis buffer into each well, and leave it at least 30 min to complete cell lysis.
6. Fill disposable 1 cm plastic cuvets with 0.75 ml of lysis buffer. Add 0.4 ml of cell lysate into the plastic cuvet and dilute it with another 0.75ml of lysis buffer, so that the final volume is 1.9 ml.
7. Measure the fluorescence of lysate in a spectrofluorometer (SLM/Aminco 500C). Fluorescein can be measured at excitation (exc.) 495 nm, emission (em.) 514 nm. Lucifer yellow is exc. 430 nm, em. 580 nm. These wavelength allowed 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, em. 540 nm.
8. Measure the protein concentration of lysates remaining in wells using a BCA protein assay kit (Pierce Chemical Co.).
9. Prepare the standard solutions, 0, 1, 5, 10, 20 ng probe/ml in lysis buffer and measure in the spectrofluorometer to obtain a standard curve.
10. Calculate the amount of probes from the standard curve and express the value as ng probe/mg protein.
IV. Pitfalls
1. A prolonged exposure to intense excitation light may cause 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.
2. Lucifer yellow can be seen using some fluorescein filter sets with wide band pass (eg. Olympus BP490), but not some others (eg. Zeiss No.09). Choose an appropriate filter set for lucifer yellow (eg. Omega Optical Set No. XF-14, Zeiss No.05).
3. Many kinds of fluorescent conjugated probes are commercially available; however, some are not good fluid-phase probes. Texas red albumin is very efficiently taken up by adsorptive endocytosis, although it was sometimes used as fluid-phase probes. Lysine-fixable Texas red dextran may bind nonspecifically to cells and cover slips.
4. For quantitative analysis, plate the cells at higher density to increase the sensitivity for fluorescence.
References
Araki, N., Johnson, M.T., Swanson, J.A. (1996) A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. J. Cell Biol 135, 1451-1460
Berthiaume, E.P. , Mediana, C. and Swanson, J.A. (1995) Molecular size-fractionation during endocytosis in macrophages. J. Cell Biol. 129, 989-998.
Racoosin, E.L. and Swanson, J.A. (1994) Labeling of endocytic vesicles using fluorescent probes for fluid-phase endocytosis. "Cell Biology: A Laboratory Handbook" (J.E. Celis ed.) pp. 375-380, Academic Press,
Racoosin, E.L. and Swanson J.A. (1993) Macropinosome maturation and fusion with tubular lysosomes in macrophages. J. Cell Biol. 121, 1011-1020.
Swanson, J.A. (1989a) Phorbol esters stimulate macropinocytosis and solute flow through macrophages. J. Cell Sci. 94, 135-142
Swanson, J.A. (1989b) Fluorescent labeling of endocytic compartments. In "Methods in Cell Biology, Vol. 29: Fluorescence Microscopy of Living Cells in Culture: Part A" (Y.-l. Wang and D. L. Taylor, eds.), pp. 137-151, Academic Press, New York
Swanson, J.A. and Watts, C. (1995) Macropinocytosis. Trends Cell Biol 5, 424-428.
Key Words endocytosis; endosomes; lysosomes; fluorescence; macrophages Figure
Legends
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, then briefly washed and fixed immediately. A low molecular weight probe, lucifer yellow, mol. wt. 457 labels both macropinosomes and micropinosomes (A). A larger probe, FDx150, mol. wt. 150,000, labels predominantly macropinosomes (B). Bars: 10um
Figure 2. Immunofluorescence of cathepsin D in endocytic compartments labeled with endocytic probes. Macrophages were incubated for 30 min in labeling medium containing 0.5 mg/ml lysine-fixable FDx10 and chased for 30 min. Then cells were fixed and processed for immunofluorescence using a primary antibody against cathepsin D and Texas red-conjugated secondary antibody. (A) Fluorescein image shows that FDx10 labels tubular lysosomes. (B) Texas-red image shows that tubular lysosomes, which are labeled with FDx10, are positive for cathepsin D. Bar: 10 µm