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. Author manuscript; available in PMC: 2026 Jun 2.
Published in final edited form as: Methods Enzymol. 2009;452:383–402. doi: 10.1016/S0076-6879(08)03623-9

Kinetic Analysis of Autophagosome Formation and Turnover in Primary Mouse Macrophages

Michele S Swanson 1, Brenda G Byrne 1, Jean-Francois Dubuisson 1
PMCID: PMC13224749  NIHMSID: NIHMS2173134  PMID: 19200894

Abstract

Macrophages enlist autophagy to combat infection by a variety of bacteria, viruses, and parasites. In response to this selective pressure, some pathogenic microbes have acquired strategies to evade or tolerate autophagy. Accordingly, infected cells may accumulate numerous autophagic vacuoles/autophagosomes when microbial products either stimulate their formation or inhibit their maturation. To distinguish between the two mechanisms, we describe methods to assess the impact of infection on the kinetics and amplitude of autophagosome formation and maturation within mouse macrophages by microscopy or Western analysis using antibodies specific for endogenous or recombinant LC3 protein.

1. Introduction

In addition to its roles in morphogenesis, cellular differentiation, and tissue remodeling, autophagy combats infection (Kirkegaard et al., 2004; Swanson, 2006; Levine and Deretic, 2007). Its contribution to immunity became apparent when, after activation by cytokines, mouse endothelial cells engulfed and digested cytosolic Rickettsia conorii within autophagic vacuoles (Walker et al., 1997). Likewise, activated macrophages also degrade vacuolar M. tuberculosis and T. gondii by autophagy (Gutierrez et al., 2004; Andrade et al., 2006).

The innate immune system has also co-opted this ancient membrane-trafficking pathway. When certain extracellular microbe-associated molecular patterns (MAMPs) are detected by their cognate toll-like Receptors (TLRs; Kawai and Akira, 2006), macrophages induce autophagy (Xu et al., 2007; Sanjuan et al., 2007; Delgado et al., 2008). Autophagy also sequesters and degrades a variety of bacteria that invade the cytoplasm, including some Group A Streptococcus, Francisella tularensis, Salmonella enterica, and Listeria monocytogenes, which escape into the cytosol (Py et al., 2007; Checroun et al., 2006; Birmingham et al., 2006; Birmingham et al., 2008; Rich et al., 2003). Several pathogens that persist within vacuoles also encounter the autophagy pathway when their specialized secretion systems deliver virulence factors into the cytoplasm. For example, S. enterica induces autophagy when its type III secretion system perforates the phagosomal membrane (Birmingham and Brumell, 2006), whereas L. pneumophila, B. abortus, and likely C. burnetii each need type IV secretion for sustained interactions between their phagosomes and the autophagic machinery (Swanson and Isberg, 1995; Celli et al., 2003; Amer et al., 2005; Romano et al., 2006). In each case, mutant bacteria that fail to breach their phagosomal membranes do not activate the autophagy machinery. Accordingly, autophagy also appears to be activated by a host surveillance system that monitors the cytosol. Indeed, both human and mouse studies implicate nucleotide oligomerization domain-like receptors (NLRs; Wilmanski et al., 2008) as regulators of autophagy. For example, the amount of the mouse NLR protein Naip5 correlates not only with robust proinflammatory death but also with the speed of autophagosome maturation and delivery of L. pneumophila to lysosomes (Amer et al., 2005). The mouse macrophage response to S. flexneri also reveals a regulatory link between pyroptosis and autophagy (Suzuki et al., 2007). Thus, host cells may elevate autophagy as a barrier to infection when pathogens either invade the cytosol or deposit in the cytoplasm virulence factors tainted with MAMPs (Swanson and Molofsky, 2005; Swanson, 2006).

Genetic studies of a human inflammatory bowel disease also point to a role for NLR proteins as critical regulators of autophagy that modulate the inflammatory response to microbial products. Humans with Crohn’s disease suffer chronic inflammation, apparently due to a dysregulated response to the gastrointestinal tract flora. For some patients, the disease is conferred by a mutant NLR protein, NOD2 (Hugot et al., 2001; Ogura et al., 2001); others encode a variant of the autophagy gene Atg16L1 (Parkes et al., 2007; Rioux et al., 2007). Accordingly, it is urgent that methods be developed to allow researchers to exploit both mouse and bacterial genetics to identify the host and microbial factors that equip macrophages to recruit the autophagy pathway as an arm of the acquired and innate immune systems.

The observation that extracellular or intracellular microbes or MAMPs can trigger accumulation of large numbers of autophagosomes in macrophages can be interpreted in two different ways: Infection either stimulates autophagosome formation or inhibits autophagosome maturation (Fig. 23.1). To distinguish between the possibilities, it is necessary to measure flux through the pathway (Klionsky et al., 2008; Mizushima and Yoshimori, 2007; Tanida et al., 2005). However, these experiments present technical challenges: in primary macrophages, autophagosomes are short lived, and reagents to label these organelles are limited. To facilitate kinetic and quantitative studies of the impact of infection on the autophagy pathway of mouse macrophages, we have developed a sensitive and specific assay that exploits LC3 to analyze the maturation of a large, synchronous population of preformed autophagosomes.

Figure 23.1.

Figure 23.1

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The number of autophagosomes in a cell is determined by the rates of both formation and maturation. When the constitutive rate of autophagy (1, box) is stimulated, the number of LC3-GFP+ vacuoles in cells increases (2, box). When the rate of autophagosome maturation is slower than normal, the number of vacuoles also increases (3, box).Whether a bacterial or host factor stimulates or inhibits the autophagy machinery can be deduced from time-course experiments performed in the absence or presence of reagents known either to stimulate autophagosome formation or to inhibit their progression to autolysosomes.

2. Monitoring Flux Through the Autophagy Pathway

To induce autophagosome formation, primary macrophages derived from the bone marrow of mice are incubated in Hank’s buffer, which lacks amino acids. After incubation for 5–55 min, the macrophages are fixed, autophagosomes are stained fluorescently, and then the number of autophagosomes in each cell is quantified. Under these conditions, within 25 min the majority of C57B1/6 mouse macrophages contain multiple large autophagosomes (Fig. 23.2), organelles easily identified by their decoration with LC3, a mammalian homolog of the yeast autophagy-related (Atg) protein Atg8 (Yamamoto et al., 2001; Kabeya et al., 2000) and the most stable and specific component of autophagosomes identified to date (Klionsky et al., 2008).

Figure 23.2.

Figure 23.2

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Primary mouse macrophages respond rapidly to autophagy stimulants. Macrophages derived from the bone marrow of GFP-LC3 transgenic C57BL/6 mice were incubated with Hank’s buffer for the periods shown, fixed with PLP-sucrose, stained with GFP-specific primary antibody (1:250 dilution; Roche, #11814460001) and Oregon Green-conjugated secondary antibody (1:1000 dilution; Invitrogen, #O-6383), and viewed by fluorescence microscopy (A), and then the fraction of macrophages containing >3 GFP-LC3+ vacuoles was scored (B).

In macrophages, autophagosomes rapidly merge with lysosomes, wherein both the contents and the LC3 protein are degraded. To determine how quickly the LC3 autophagosomal marker is extinguished within lysosomes in primary mouse macrophages, cells are subjected to starvation for 30 min and then transferred to rich medium. Within 30 min, the population of LC3 autophagosomes is diminished to baseline levels (Fig. 23.3).

Figure 23.3.

Figure 23.3

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Autophagosomes mature quickly in primary mouse macrophages. To generate autophagosomes, C57B1/6 GFP-LC3 transgenic macrophages were subjected to amino acid starvation by incubating for 30 min with Hank’s buffer. To measure the rate of autophagosome maturation, cells were then fed (chase: RPMI). To determine the maximal and minimal autophagy response, macrophages were continuously starved (no chase), or fed (no starvation), respectively. At the times shown, the fraction of macrophages containing >3 GFP-LC3+ vacuoles was quantified by microscopy.

To verify that fusion of autophagosomes with lysosomes accounts for the extinction of the GFP-LC3 signal, vacuole maturation is inhibited pharmacologically (Tanida et al., 2005; Mizushima and Yoshimori, 2007). After stimulating autophagy by starving macrophages for 40 min, the pH of the endosomal compartment is neutralized by incubating the cells for another 20 min with the proton pump inhibitor bafilomycin A1 (Yamamoto et al., 1998). Indeed, when progression to lysosomes is blocked, the fraction of cells that contain >5 GFP-LC3+ vacuoles increases from 19 to 59% (Fig. 23.4).

Figure 23.4.

Figure 23.4

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The GFP-LC3 signal persists when macrophage proton pumps are inhibited with bafilomycin A1, which retards the progression of autophagosomes to autolysosomes. To stimulate autophagosome formation, macrophages derived from the bone marrow of C57BL/6 mice were incubated in Hank’s buffer for 40 min. To measure the impact of vacuole acidification on autophagosome maturation, the macrophages were incubated for an additional 20 min in the presence or absence of bafilomycin A1. The fraction of macrophages that contained 0 (white), 1–5 (gray), or >5 (black) autophagic vacuoles/autophagosomes was quantified by fluorescence microscopy after amplifying the GFP-LC3 signal with GFP-specific antibody (Roche).

To verify results obtained by microscopy, the kinetics of activation and turnover of LC3 is assessed by Western analysis (Mizushima and Yoshimori, 2007; Tanida et al., 2005). When activated, LC3 is covalently linked directly to phosphatidylethanolamine, a modification that speeds migration of the enzyme during SDS-PAGE (Tanida et al., 2005). Accordingly, the kinetics and amplitude of LC3 activation can be assessed by Western analysis of lysates obtained from macrophages subjected to a pulse of starvation. Control macrophages contain primarily nonlipidated LC3, whereas starved cells also contain the lipidated protein (LC3-II), the form that is preferentially recognized by an antibody available from Novus Biologicals (Fig. 23.5). If the rapid loss of GFP-LC3 antigenicity detected by fluorescence microscopy reflects robust fusion with lysosomes, expect to observe by Western analysis a progression from nonlipidated LC3 to lipidated LC3 (LC3-II), to a decline of lipidated protein (Mizushima and Yoshimori, 2007). In addition, expect treatment of cells with pharmacological inhibitors of either the proton pump or lysosomal proteases to stabilize the lipidated LC3 (LC3-II; Mizushima and Yoshimori, 2007; see also the chapter by Kimura et al., in this volume).

Figure 23.5.

Figure 23.5

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Kinetic analysis of lipidation and turnover of endogenous LC3 protein by mouse macrophages using a commercial antibody. To induce a synchronous pulse of autophagy, C57Bl/6 mouse macrophages were incubated in rich medium (RPMI) or amino acid-free Hank’s buffer (Hank’s) for the periods shown before transfer to rich medium for the times indicated. Lysates of 106 cells were separated by SDS-PAGE on a 4%–20% gradient acrylamide gel, transferred to membrane, probed with rabbit polyclonal anti-LC3 (Novus Biologicals, #NB 100-2331) and an HRP-conjugated secondary antibody (Santa Cruz Biotechnology, sc-2030), and then developed to visualize the activated LC3-II protein. Size markers indicate that LC3-II is approximately 14 kDa, and a nonspecific band of approximately 50 kDa indicates that each lane contains a similar quantity of protein (not shown).

3. Assessing the Impact of Infection on the Autophagy Pathway

To test whether a particular microbe stimulates flux through the autophagy pathway, starved macrophages are transferred to rich medium that contains or lacks the microbe of interest, in our case L. pneumophila. To gauge the specificity and sensitivity of these assays, we assess in parallel the impact of virulent and mutant bacteria on autophagosome maturation. For example, macrophages are subjected to starvation by incubating with Hank’s buffer for 20 min, and then the cells are incubated for 5–40 min with either wild-type L. pneumophila or dotA mutant bacteria, which are defective for type IV secretion. Even within 5 min of infection, it is evident that L. pneumophila stimulates autophagosome maturation, as the number of cells that contain GFP-LC3+ vacuoles declines at a rate markedly faster than that observed for uninfected macrophages (data not shown). After 40 min of infection with virulent bacteria, macrophages are devoid of autophagosomes, as judged by their loss of GFP-LC3 antigenicity (Fig. 23.6). Moreover, rapid turnover of the autophagosomes is triggered by either the activity or a substrate(s) of the type IV secretion apparatus, as macrophages retain numerous autophagosomes, similar to uninfected macrophages, when they harbor motile bacteria that are defective in type IV secretion (dotA mutants). Thus, in response to a microbe equipped to pierce its phagosome and deliver microbial products to the cytosol, macrophages derived from the bone marrow of C57BL/6 mice stimulate rapid autophagosome maturation.

Figure 23.6.

Figure 23.6

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Type IV secretion-competent Legionella stimulate autophagosome maturation. GFP-LC3 transgenic macrophages were treated for 20 min with Hank’s buffer, cultured for 40 min with wild-type or mutant bacteria as shown, fixed with PLP-sucrose, stained with GFP-specific primary (1:250 dilution; Roche) and Oregon Green-conjugated secondary antibody (1:1000 dilution; Molecular Probes), and viewed by fluorescence microscopy (A). Arrows indicate infected cells. The fraction of macrophages that contained >3 GFP-LC3+ vacuoles (black), bacteria only (white), or both (gray) was scored (B).

4. LC3 localization in Primary Mouse Macrophages

In primary mouse macrophages, LC3 can be localized by one of four methods. Mizushima and colleagues have generated and characterized a transgenic line of C57BL/6 mice that encode recombinant GFP-LC3 (Mizushima et al., 2004; see also the chapter by N. Mizushima in this volume). By using macrophages derived from the bone marrow of this mouse strain, investigators are assured that every cell expresses similar amounts of GFP-LC3 while avoiding artifactual LC3 aggregation due to overexpression (Klionsky et al., 2008). With our equipment, it is beneficial to amplify the signal using a GFP-specific primary antibody (Roche, #11814460001) and a fluorophore-conjugated secondary antibody (Invitrogen, Oregon Green #O-6383).

As an alternative to monitoring recombinant transgenic GFP-LC3, a polyclonal antibody that is specific to LC3 and suitable for detecting the endogenous protein in mouse macrophages by immunofluorescence microscopy is currently available from MBL International Corporation (#M152-3; Fig. 23.7). To analyze the impact of particular mouse mutations on macrophage autophagy, this is currently the best option.

Figure 23.7.

Figure 23.7

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A similar response is observed when autophagosomes are labeled via endogenous LC3 or the GFP-LC3 transgene. Macrophages derived from the bone marrow of commercial (A, C, D) or transgenic GFP-LC3 (B) C57Bl/6 mice were incubated for 25 min in rich medium (A, RPMI) or Hank’s buffer (B-D), then autophagosomes were visualized using antibody specific to GFP (1:250 dilution, Roche; B) or an LC3 antibody prepared commercially (1:150 dilution, MBL; C), or by a laboratory protocol (Taylor and Kirkegaard, 2007; D).

The Kirkegaard laboratory has also published a protocol to generate polyclonal antibodies against recombinant human LC3 that cross-reacts with the mouse protein (Taylor and Kirkegaard, 2007). Antibody quality is evaluated by comparing sera obtained from multiple rabbits before and after immunization using Western analysis of macrophage lysates as the initial screen, and localization of LC3 in fed and starved macrophages as the secondary screen. Note that some antibody preparations are suitable for Western analysis but not immunofluorescence microscopy. Using antibody prepared by this protocol, endogenous LC3 protein is readily detected on autophagosomes formed when primary C57B1/6 macrophages are subjected to amino acid starvation (Fig. 23.7.)

Another general strategy is to transduce mammalian cells with recombinant LC3 protein. For this purpose, a newly developed specific probe, tandem-tagged fluorescent LC3 (tflLC3; Kimura et al., 2007; see also the chapter by Kimura et al., in this volume) offers an important advantage: the ability to monitor progression of autophagosomes to autolysosomes. Recombinant mRFP-GFP-LC3 protein exploits the observation that GFP is degraded and therefore quenched in acidic lysosomes, whereas RFP is relatively stable (Mizushima and Yoshimori, 2007). Therefore, in mammalian cells, immature autophagosomes are visible in both the red and green channels, but acidic autolysosomes fluoresce only red. The primary drawbacks of using recombinant proteins to analyze autophagy are the time and expense required to introduce the transgene either into the cells or the animals of each genotype of interest. In transfection experiments, cell-to-cell variability in expression and artifactual patterns due to aggregation of the recombinant protein can also be problematic (Klionsky et al., 2008).

5. Isolation of Bone Marrow-Derived Macrophages

This method is adapted from Celada et al. (1984) and typically yields after 1 week in culture 8 × 107 macrophages per mouse.

5.1. Prepare media

L-cell supernatant: L-cells are a mouse fibroblast cell line that secretes Macrophage Colony Stimulating Factor (M-CSF). Culture L-cells in RPMI + 10% FBS-heat inactivated + pen/strep. A subconfluent culture of L-cells can be used to seed flasks; alternatively, thaw a fresh aliquot of cells stored in liquid nitrogen for each supernatant preparation. The shelf life at −70 °C is at least 6 months; at −20 °C, 2–3 months; at 4 °C, only 2–3 weeks.

  1. Transfer from liquid nitrogen storage an aliquot of L-cells to a 37 °C bath, then resuspend the thawed cells in 9 ml of 37 °C medium.

  2. Collect the cells by centrifugation at 250 × g for 10 min.

  3. Aspirate the supernatant fraction and resuspend the cell pellet in 6 ml of medium.

  4. Incubate in a 25-cm2 flask overnight at 37 °C and 5% CO2.

  5. Wash away debris with warm medium and replace with 6 ml of fresh medium.

  6. When confluent, trypsinize the monolayer: Remove the medium, add 5 mL of 0.05% Trypsin-EDTA (Gibco #25300) and let stand at RT for 5 min. Tap the flask and pipette up and down gently a few times to release any remaining attached cells. Transfer the cell suspension to a 15-mL conical tube and collect by centrifugation at 250 × g for 10 min at 4 °C. Remove the supernatant fraction and resuspend the cell pellet in 8 mL of RPMI containing 10% FBS. Count the cells using a hemacytometer.

  7. Replate 3 × 105 cells in 60 ml of medium per 150-cm2 flask.

  8. Incubate until there is a confluent monolayer (approximately 5 days).

  9. Collect the supernatant: Pour the conditioned medium through a bottle-top 0.22-μ filter into sterile bottles.

  10. Freeze aliquots at −20 °C, then store at −70 °C.

Bone marrow macrophage culture medium: For each mouse, prepare 100 ml in RPMI.

30% L-cell supernatant

1% Penicillin-Streptomycin (pen/strep; Gibco, #15140)

20% FBS, heat-inactivated (Gibco, #10437-028; To heat inactivate complement proteins, thaw the bottle of FBS overnight at 4 °C, heat to 56 °C for 30 min, and store aliquots at −20 °C)

5.2. Setup

  1. Chill to 4 °C:
    • 10-ml syringe equipped with 25-gauge needle and filled with 10 ml of RPMI
    • 2 15-ml tubes
    • Centrifuge
  2. Have ready sterile:
    • Forceps
    • Scissors
    • Scalpel
    • 100-mm Petri plates

5.3. Dissection to obtain both femurs and tibias

  1. Sacrifice 1 mouse.

  2. Pin the mouse spread-eagle with the abdomen facing up on the dissecting tray.

  3. Sterilize the mouse abdomen with 70% ethanol.

  4. Using forceps, lift the skin at the hip joint away from the animal, then snip with scissors.

  5. Peel the skin back to expose the quadriceps muscle from the hip to the knee joint.

  6. Using the forceps and scalpel, remove each leg above the hip.

  7. Using the forceps to grasp the quadriceps muscle, detach to expose each femur and tibia.

  8. Using the forceps, detach each femur by cutting with the scalpel above the proximal ball joint and below the distal ball joint. Repeat for the second femur.

  9. Using the scalpel, scrape the connective tissue from the femurs and tibias.

  10. Wash the femurs and tibias with 70% ethanol.

5.4. Collect bone marrow cells

  1. Move the femurs and tibias, the sterilized scissors and the forceps into a sterile hood.

  2. Cut both ball joints from one femur.

  3. Insert the needle into the hollow of the femur and pulse with 2.5 ml of medium, flushing the marrow cells into the tube on ice. Repeat from the other end of the femur.

  4. Repeat with the other femur and both tibias.

  5. Release cells from the marrow by gentle pipetting with a 10-ml pipette and by inverting the closed tube several times.

  6. Allow large flocculent material to settle to the bottom of the tube by standing on ice for 5 min.

  7. Place 3–4 × 106 cells in approximately 15 ml of bone marrow macrophage medium in each 100-mm Petri dish. Typically, if the cell harvest went smoothly, we divide the cells evenly among 5 dishes. Do not use tissue-culture grade plastic, as the tightly adherent mature macrophages will be difficult to dislodge. Instead, we use deep Petri dishes, 100 × 25 mm (Nunc, Lab-Tek #4031).

  8. On day 3, feed the cells by adding approximately 5 ml of bone marrow macrophage medium.

5.5. Replate macrophages for experiments

On day 6 or 7, replate adherent cells to tissue culture wells or coverslips. Because primary macrophages readily adhere to tubes and pipettes, best yields are obtained when reagents and tissue culture supplies are ice cold.

  1. Aspirate the supernatant and add approximately 10 ml of ice-cold PBS that lacks divalent cations (Gibco, #10010) to inhibit adherence mediated by integrin receptors.

  2. Let dish stand on ice for approximately 5 min to promote cell detachment.

  3. Using gentle pipetting, flush the monolayer from the dish.

  4. Transfer the suspension to an ice-cold 50-ml conical tube.

  5. Wash the dish with an additional 5–10 ml of ice-cold PBS.

  6. Collect the cells by centrifugation at 400 × g for 10 min at 4 °C, and discard the supernatant fraction by decanting or aspiration.

  7. Resuspend the cells in 10 ml of 10% FBS in RPMI (Gibco, #11875) and count using a hemacytometer, expecting 12–18 × 106 macrophages per dish.

  8. Adjust to the desired concentration in 10% FBS in RPMI (see subsequently) and plate for experiments. For studies of the impact of bacteria on macrophage biology, omit antibiotics.

Cells attach and spread within a 30-min incubation at 37 °C and 5% CO2. In our experience, cells can be used after approximately 90 min or within the next 4–5 days (the period varies with mouse genotype). With time, the cells grow larger and more thinly spread.

5.6. Cell densities

  • 2.5 × 105 macrophages per 0.5 ml per well of a 24-well plate will form a confluent monolayer for bacterial growth curve studies.

  • 106 macrophages per 35 mm well of a 6-well plate for Western analysis.

  • 7.5 × 104 to 105 macrophages per glass coverslip (#1 thickness, Fisher Scientific, #12-545-80) for microscopy.

5.7. Quantification of autophagosome dynamics by immunofluorescence microscopy

  1. Culture 7.5 × 104 to 105 macrophages per 0.5 ml of RPMI + 10% FBS per glass coverslip (#1 thickness, Fisher Scientific, #12-545-80) in 24-well plates.

  2. To induce a pulse of autophagy, aspirate the medium and replace with 0.5 ml of Hank’s buffer (see subsequently) prewarmed to 37 °C and incubate at 37 °C for the desired period. (For bone marrow–derived macrophages from C57B1/6 mice, the maximum number of autophagosomes is observed after approximately 25 min of incubation with Hank’s buffer; Fig. 23.2.)

  3. Option A: To allow the population of autophagosomes to mature, aspirate Hank’s buffer and replace with RPMI/FBS prewarmed to 37 °C. (For bone marrow–derived macrophages from C57B1/6 mice, the number of autophagosomes returns to baseline after approximately 20 min incubation with RPMI/FBS; Fig. 23.3.)

    Option B: To investigate the impact of microbes or microbial products on autophagy, replace Hank’s buffer with RPMI/FBS that contains or lacks the microbe or product of interest at an optimal concentration, determined empirically, and then incubate for the desired period(s), also determined empirically (e.g., Fig. 23.6).

  4. Replace Hank’s buffer or RPMI/FBS with freshly prepared modified PLP-sucrose (see subsequently) prewarmed to 37 °C for 30 min at room temperature.

  5. Wash 3 times with PBS.

  6. Methanol extract to permeabilize the membranes: Using a fine-tip forceps, gently swish each coverslip for 5 s in −20 °C methanol held on ice.

  7. Rinse 3 times with PBS.

  8. Block 5 min at RT with PBS + 2% heat-inactivated goat serum (PBS/GS; Gibco #16210-064). To heat-inactivate complement components in the serum, heat in a water bath to 56 °C for 30 min; store aliquots at −20 °C.

  9. Incubate overnight at 4 °C with the appropriate dilution of primary antibody in PBS/GS. To minimize the volume of antibody needed, prepare an incubation chamber: invert a 24-well tissue culture plate, stretch Parafilm across the solid bottom surface, and place 25–50 μL drops of diluted antibody onto the Parafilm, using the well outlines as a guide. Using a fine-tip forceps, remove the coverslip from the well and invert onto a drop of antibody; repeat for all the samples. Cover the coverslips using the top of the 24-well culture plate. At the end of the overnight incubation, transfer each coverslip, right side up, back into the original 24-well plate for washes.

  10. Wash 3 times for 5 min with PBS/GS.

  11. Incubate 1 h at 37 °C in a humidified incubator with 25–200 μl of the appropriate dilution of secondary antibody in PBS/GS.

  12. Wash 3 times for 5 min with PBS.

  13. Remove each coverslip from its well, gently wipe the PBS off the back of the coverslip, then place the coverslip cell-side-down onto a tiny drop of mounting medium (Prolong Gold with DAPI, Invitrogen/Molecular Probes, #P369935) on a plain glass microscope slide (25 × 75 × 1 mm). Aspirate excess mounting medium that seeps out from the coverslip. Let dry overnight at RT protected from light.

  14. Add a tiny drop of immersion oil and observe fluorescence using the appropriate filter sets.

5.8. Controls

  1. To gauge the background level of autophagy, fix one untreated sample.

  2. To gauge nonspecific staining by the secondary antibody, omit the primary antibody from one sample.

  3. To determine the appropriate dilution of the primary antibody, analyze a series of dilutions (e.g., 1:50, 1:100, 1:250) using a constant secondary antibody concentration. Choose the dilution that yields the optimal signal:noise ratio.

  4. To determine the appropriate dilution of secondary antibody, analyze a series of dilutions (e.g., 1:100, 1:200, 1:400) using a constant primary antibody concentration and a control lacking primary antibody. Choose the dilution that yields the optimal signal:noise ratio.

  5. As a guard against physical damage or loss during handling, use duplicate coverslips for each sample.

  6. To determine the specificity of the response to the microbial product of interest, analyze the appropriate controls in parallel (e.g., isogenic mutant that lacks product, mutant protein that lacks activity, formalin-killed microbes, denatured wild-type protein, or the like).

5.9. Hank’s buffer

To induce autophagy, macrophages are transferred to Hank’s Balanced Salt Solution (Gibco, #14025), which lacks amino acids.

Note: Be certain the buffer contains calcium, which is essential for a macrophage autophagy response.

5.9.1. Periodate-lysine-paraformaldehyde fixative (PLP) + 5% sucrose

This fixative has been modified from McLean and Nakane (1974) to include sucrose as an osmotic support for fragile membrane networks, including the endosomal compartment and the endoplasmic reticulum. The final solution contains 10 mM NaIO4, 75 mM lysine, 37.5 mM NaPO4, 2% paraformaldehyde, and 4.5% sucrose.

40 mls Stock A

20 ml 0.2 M Lysine

15.5 ml 0.1 M Na2HPO4

4.5 ml 40% sucrose

Store at 4 °C.

8% Paraformaldehyde

  1. Weigh out 1–2 g of paraformaldehyde (toxic: wear gloves, use mask, weigh in chemical fume hood).

  2. Add water to bring up to 8%.

  3. Heat with stirring in a fume hood to approximately 70 °C.

  4. Add a drop of 1 N NaOH; the solution should clear.

  5. When cool enough to handle, filter to remove particles.

  6. Store at 4 °C and protected from light by wrapping in foil.

  7. For best results, use on the day of preparation. Alternatively, store up to approximately 1 month at −20 °C as 2.5-ml aliquots in 15-ml amber disposable tubes; thaw and prewarm to 37 °C until the solution clears before use.

5.9.2. 10 ml PLP-sucrose fixative

Dissolve 21.4 mg of sodium periodate (toxic: wear gloves) in 7.5 ml of Stock A and 2.5 ml of 8% paraformaldehyde. For best results (i.e., preservation of endoplasmic reticulum or endosomal network) make fresh and warm to 37 °C before use.

5.10. Western analysis of lipidated LC3

In our experience, the limit of detection for endogenous LC3 protein per lane requires a protein extract from 106 bone marrow–derived mouse macrophages. The LC3 protein is also labile: even during storage of boiled protein samples overnight at −20 °C, the signal deteriorates.

  1. Culture 106 macrophages per well of a 6-well tissue culture plate (e.g., Costar #3516).

  2. To assess activation of autophagy, aspirate the culture medium, replace with Hank’s buffer (Gibco, #14025), and incubate in a humidified incubator at 37 °C and 5% CO2 for the desired periods. (For C57Bl/6 macrophages, we observe peak LC3-II approximately 35 min after exposure to Hanks’s buffer; Fig. 23.5.)

  3. To assess maturation of autophagosomes, treat macrophages with a pulse of Hank’s buffer for the period determined empirically to generate maximal levels of the LC3-II protein, then replace Hanks’s buffer with prewarmed RPMI + 10% FBS for the desired periods. (For C57B1/6 macrophages, we observe that LC3-II has diminished to baseline levels approximately 20 min after a return to rich medium; Fig. 23.5.)

  4. Rinse monolayers twice with 4 °C PBS, then add 1 ml of cold PBS/well.

  5. Dislodge the cells using a Disposable Cell Scraper (Fisher, #08-773-2) and transfer the cell suspension to an ice-cold microcentrifuge tube.

  6. Repeat for each sample, keeping all of the cell suspensions on ice.

  7. Collect the cells by centrifugation for 10 min at 5000 rpm in a microcentrifuge.

  8. Aspirate the supernatant fraction and resuspend the cell pellet in 15 μl of ice-cold PBS.

  9. Lyse the cells by addition of 15 μl of 2X Laemmli sample buffer and boil in a water bath for 5 min.

  10. Clarify the sample by centrifugation for 5 min at 10,000 rpm in a microcentrifuge.

  11. Load 30 μl of each sample (equivalent to 106 macrophages) per lane of either a 4%–20% linear gradient SDS-polyacrylamide gel (Fig. 23.5) or a 15% SDS-polyacrylamide gel, and separate by electrophoresis using a constant 70 V.

  12. Using a semidry blotting system, transfer to PVDF membranes (Bio-Rad) by applying 15 V for 45 min.

  13. Block the membranes by incubating overnight at 4 °C in 5% dry milk and 0.1% Tween 20 in Tris-buffered saline (TBS, Bio-Rad, #170-6435).

  14. To detect endogenous protein, incubate with LC3-specific rabbit antibody (1:500 dilution; Novus Biological, #NB 100-2331) for 1 h at RT with agitation on a platform shaker. Alternatively, to detect recombinant GFP-LC3 protein, incubate with GFP-specific mouse antibody (Roche, #11814460001; 1:1000 dilution).

  15. Wash with TBS + 0.1% Tween 20, 5 times, 5 min per wash, at RT.

  16. Incubate with HRP-conjugated anti-Rabbit IgG secondary antibody (1:3000 dilution; Santa Cruz Biotechnology, sc-2030). Alternatively, to detect recombinant GFP-LC3 protein, incubate with peroxidase-conjugated mouse IgG-specific secondary antibody (Sigma, A9917, 1:4,000) for 1 h at RT with agitation on a platform shaker.

  17. Wash with TBS + 0.1% Tween 20, 5 times, 5 min per wash, at RT with agitation on a platform shaker.

  18. Detect specific protein using the enhanced chemiluminescence protocol described by the manufacturer (Pierce; Supersignal West Pico).

6. Conclusion

Our autophagy assay offers several technical advantages. By first subjecting macrophages to amino acid starvation, we generate a large and synchronous population of autophagosomes in the absence of confounding MAMPs, ubiquitous microbial products likely to affect the pathway (Delgado et al., 2008; Sanjuan et al., 2007; Xu et al., 2007). By restricting our infection and assay periods to <1 h, we limit the impact of developmental changes by either the macrophages or the microbes. Rapid effects of bacterial factors on turnover of GFP-LC3+ vacuoles can be quantified within single cells by microscopy. Alternatively, autophagosome formation and flux can be assessed by Western analysis. Thus, using the protocols provided here and reagents now available, it is possible to analyze the impact of microbes or their products on the amplitude and kinetics of autophagosome formation and maturation within primary macrophages derived from the bone marrow of wild-type or mutant mice.

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