Abstract
Macrophages play a key role in the innate immune response and help to direct the acquired immune response. Early in the innate immune response, they produce reactive oxygen species and pro-inflammatory cytokines and chemokines to drive inflammation and are referred to as “classically activated” or “killer” macrophages (M1). During the resolution phase of inflammation, they switch to what is known as an “alternatively activated” phenotype or “healer” macrophage (M2) and contribute to debris scavenging, angiogenesis, and wound healing. M1 macrophages are activated by treatment with IFNγ or LPS and M2 macrophages are activated by treatment with Th2 cytokines IL-4 or IL-13 and the M2 phenotype switch can be enhanced by IL-10. Macrophages can also be skewed during differentiation in vitro, and the resultant phenotype depends upon the cytokine provided to support their differentiation. In murine macrophages, MCSF promotes differentiation to an M1 phenotype, GM-CSF promotes differentiation to an M2 phenotype and IL-3 promotes differentiation into a profoundly M2 skewed phenotype. A defining feature of the phenotype of murine M1 versus M2 macrophages is how they metabolize L-arginine. In response to an inflammatory stimulus like LPS, M1 macrophages produce inducible nitric oxide synthase (iNOS) which uses L-arginine as a substrate to produce nitric oxide (NO). M2 macrophages constitutively produce the enzyme arginase I (argI), which sequesters L-arginine from iNOS and results in the production of ornithine and downstream polyamines and L-proline. M1 macrophages also produce relatively higher levels of pro-inflammatory IL-12 and lower levels of anti-inflammatory IL-10 relative to M2 macrophages. In this chapter, we describe in vitro derivation of polarized bone marrow macrophages and methods to analyze the resulting phenotype including Q-PCR, Western blotting, and enzyme assays to determine argI and iNOS expression and activity, as well as production of IL-12p40 and IL-10 and determination of IL-12/IL-10 ratios. Production of iNOS, NO, IL-12p40, and IL-10 are measured after treatment with LPS.
Keywords
1 Introduction
Macrophages are critical players in all aspects of the immune response to foreign pathogens and tumor cells. Resident tissue macrophages are poised to respond to infection or injury and initiate an inflammatory response to danger or pathogen associated molecular patterns. Within 24 h of insult, monocytes are recruited from the circulation and move into a site of injury or infection where they mature into classically activated macrophages, also called killer or M1 macrophages (1). These M1 macrophages amplify the innate immune response producing cytokines and chemokines. They can also present antigen to initiate the acquired immune response. When the inflammatory insult has been dealt with, macrophages remain at the scene and are converted by the local cytokine milieu to participate in the resolution phase of inflammation. These macrophages participate in debris scavenging, angiogenesis, tissue remodeling, and wound healing (2). These macrophages are called alternatively activated, healer, or M2 macrophages (3).
M1 and M2 macrophages represent extremes of macrophage polarization. Increasingly, we recognize that macrophages are heterogeneous both in their phenotype and functional responses to inflammatory stimuli (4). In complex systems, this may be due to multiple, simultaneous stimuli acting on individual cells, intermediate or transitional phenotypes, as well as the effects of populations of macrophages. Despite this, it is still critical to define features of polarized macrophages to enable comparison and categorization of macrophage phenotype and function to better understand their role in normal and pathophysiologies. M1 macrophages are activated by IFNγ or LPS and produce robust amounts of reactive oxygen species and pro-inflammatory cytokines (IL-12, TNFα, IL-23) and chemokines and murine M1 macrophages upregulate inducbile nitric oxide synthase (iNOS) to produce the reactive nitrogen species, nitric oxide (NO) (1). The canonical M2 macrophages, also referred to as M2a macrophages, are activated by IL-4 or IL-13 and their activation can be enhanced by co-treatment with IL-10. In response to inflammatory stimuli, these macrophages produce lower amounts of pro-inflammatory cytokines and higher amounts of anti-inflammatory IL-10 relative to their M1 counterparts. Additionally, murine M2 macrophages up-regulate expression of arginase I (argI), Ym1 (a mammalian chitinase), and FIZZ1 (also known as RELMα) (5).
A critical switch that defines murine macrophage activation and polarization is the way in which the cells metabolize L-arginine (6, 7). Murine M1 macrophages metabolize L-arginine by iNOS to produce NO. NO is a reactive nitrogen intermediate that can damage DNA, thereby killing foreign microorganisms or tumor cells and also causes host tissue damage. M2 macrophages metabolize L-arginine via argI to produce L-ornithine. L-ornithine is a precursor for putrescine, spermidine, and spermine production, which promote cell proliferation and tissue repair and for L-proline, which is an essential component of collagen biosynthesis required for tissue repair. In addition, when both argI and iNOS are expressed, argI sequesters L-arginine from iNOS acting as a cell intrinsic inhibitor of NO production. Finally, argI induction has been shown in one system to block transcription of iNOS in response to inflammatory stimuli providing another level of negative regulation of pro-inflammatory NO production (8).
It is important to understand the forces driving macrophage phenotype and the characteristics that define the resultant phenotype so that we can better understand the role of macrophages in normal physiology and in disease. Aberrant macrophage phenotype contributes to autoimmune and auto-inflammatory diseases as well as solid tumor growth, via tumor associated macrophages which share features with M2 macrophages (9, 10). The potential to drive select features of macrophage phenotype could provide novel targets for intervention in these pathologies as well as in treatment of infectious diseases. Techniques provided here will allow evaluation of the impact of unique experimental systems on deriving and characterizing polarized macrophages. Polarization of mature macrophages to an M2 phenotype has been described previously (11) and genetic models of polarized macrophage phenotype have also been described (12–14). Herein, we describe approaches to generate bone marrow derived macrophages comparing the impact of different cytokines available to the cells in vivo during differentiation that result in differentially skewed argI/NO metabolism and cytokine production profiles (15–17).
2 Materials
2.1 Tissue Culture
-
1.
C57BL/6 mouse (8–12 weeks old). Animals housed and sacrificed according to institutional requirements.
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2.
Iscove’s Modified Dulbecco’s Medium (IMDM).
-
3.
Fetal bovine serum.
-
4.
Recombinant murine macrophage colony stimulating factor (MCSF or CSF-1).
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5.
Recombinant murine granulocyte-macrophage colony stimulating factor (GM-CSF).
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6.
Recombinant murine interleukin-3 (IL-3).
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7.
Recombinant murine interleukin-4 (IL-4).
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8.
Monothioglycerol (MTG).
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9.
Culture medium: IMDM, 10% FCS, 150 μM MTG. Combine and filter-sterilize using low protein binding 0.2 micron filter. Store at 4°C for up to 1 month.
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10.
Cell Dissociation Buffer (Gibco-BRL, Bethesda, MA).
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11.
Lipopolysaccharide from Escherichia coli serotype O127:B8 (LPS).
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12.
Incubator set at 37°C and 5% CO2.
2.2 Quantitative PCR
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1.
RNAse-free 1.5 ml eppendorf tubes.
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2.
Sterile and RNAse-free filter tips.
-
3.
TRIzol® (Invitrogen).
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4.
Isopropanol.
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5.
75% (v/v) ethanol in diethylpyrocarbonate (DEPC)-treated water.
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6.
Oligo p(dT)20-40 (5 U per 750 ml of DEPC-treated water).
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7.
dNTPs mix (10 μM each in DEPC-treated water).
-
8.
MMLV reverse transcriptase.
-
9.
MMLV reverse transcriptase reaction buffer.
-
10.
iQ SYBR green Supermix Q-PCR master mix (2×) (Bio-Rad Laboratories, Hercules, CA).
-
11.
Primers:
-
argI forward: 5′-TTGCGAGACGTAGACCCTGG-3′
-
argI reverse: 5′-CAAAGCTCAGGTGAATCGGC-3′
-
iNOS forward: 5′-GCCACCAACAATGGCAACA-3′
-
iNOS reverse: 5′-CGTACCGGATGAGCTGTGAATT-3′
-
GUS forward: 5′-ACGTTAGCCGGGCTGCACTC-3′
-
GUS reverse: 5′-TCGGTTTGCGGTCGCGAGTG-3′
-
-
12.
NanoDrop Spectrophotometer (Thermo Scientific, Ottawa, ON, Canada).
2.3 SDS-PAGE
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1.
Laemmli’s digestion mix (LDM): 75 mM Tris–HCl pH 6.8, 7.5% (w/v) glycerol, 200 mM β-mercaptoethanol, 1.5% (w/v) bromophenol blue.
-
2.
Separating gel mix (4×): 1.5 M Tris–Cl pH 8.8, 0.4% SDS.
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3.
Stacking gel mix (4×): 0.3 M Tris–HCl pH 6.8, 0.4% SDS.
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4.
40% Acrylamide/bisacrylamide solution (37.5:1 with 2.6% C).
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5.
N,N,N′,N′-Tetramethyl-ethylenediamine (TEMED).
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6.
10% (w/v) Ammonium persulfate (APS). Freeze aliquots at −20°C for up to 3 months, thawing an aliquot for use and storing at 4°C for no more than 7 days.
-
7.
PageRuler prestained protein ladder (Fermentas Life Sciences, Thermofisher).
-
8.
Running buffer (10×): 0.25 M Trizma base, 1.9 M glycine, 1% (w/v) sodium dodecyl sulfate (SDS).
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9.
Glass plates (Fischer Scientific Co., Pittsburgh, PA).
-
10.
Bio-Rad Protean II xi Cell (Bio-Rad Laboratories Inc, Hercules, CA).
2.4 Western Blotting
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1.
Transfer buffer (10×): 0.25 M Trizma base, 1.92 M glycine, 0.5% (w/v) SDS.
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2.
Methanol.
-
3.
Immobilon-P membrane (0.45 μm pore polyvinyldifluoride (PVDF)) (Bio-Rad Laboratories, Hercules, CA).
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4.
Whatman filter paper.
-
5.
Tris-buffered saline with Tween-20 (TBST): 137 mM NaCl, 2.7 mM KCl, 25 mM Tris–Cl pH 7.4, 0.1% (v/v) Tween-20.
-
6.
Blocking buffer: 5% (w/v) bovine serum albumin fraction V (BSA), 0.02% NaN3 in TBST.
-
7.
Primary antibody buffer: 2% (w/v) BSA, 0.008% NaN3 in TBST.
-
8.
Primary antibodies: anti-arginase I (murine) (BD Biosciences, Mississauga, ON, Canada), anti-Ym1 (rabbit) (STEMCELL Technologies Inc, Vancouver, BC, Canada), GAPDH (murine) (Fitzgerald Industries, Acton, MA, USA). Each primary antibody is used at 1 in 1,000 (v/v) in primary antibody buffer.
-
9.
Secondary antibodies: anti-mouse horse-radish peroxidase (HRP), anti-rabbit HRP. Secondary antibodies are used at 1 in 10,000 (v/v) in TBST (Bio-Rad Laboratories, Hercules, CA, USA).
-
10.
Bio-Rad Trans-Blot Cell (Bio-Rad Laboratories Inc, Hercules, CA, USA).
-
11.
Enhanced chemiluminescent reagent “Western Lightning” (PerkinElmer, Waltham, MA, USA).
-
12.
Kodak X-OMAT blue film.
2.5 Arginase Assay
-
1.
Locking eppendorf tubes.
-
2.
Urea standard: 50 mM in ddH2O.
-
3.
Arginase lysis buffer: 0.1% (v/v) Triton X-100 in 25 mM Tris–Cl pH 8.0.
-
4.
10 mM MnCl2.
-
5.
0.5 M L-arginine in ddH2O, pH 9.7.
-
6.
Acid mixture: 1:3:7 (v/v/v) H3PO4 (44.6 N):H2SO4 (36 N):ddH2O.
-
7.
Colorimetric reagent: 9% (w/v) α-isonitrosopropiophenone (αISPP) in absolute ethanol.
-
8.
Bio-Rad protein quantification assay (Bio-Rad, Hercules, CA, USA).
2.6 NO Assay
-
1.
100 mM NaNO2: 0.69 mg NaNO2 in IMDM, 10% (v/v) FBS.
-
2.
Sulfanilamide (H2NC6SO2NH2) solution: 1% (w/v) in 2.5% (v/v) phosphoric acid (H3PO4).
-
3.
Naphthylethylenediamine dihyrochloride (C10H7NHCH2CH2NH2-2HCl-MeOH) solution: 0.1% (w/v) in 2.5% (v/v) H3PO4.
2.7 Enzyme-Linked Immunosorbent Assays
-
1.
ELISA kits for IL-12p40 and IL-10 (BD Biosciences).
-
2.
Coating buffer: 0.2 M sodium phosphate pH 6.8.
-
3.
Assay diluent: 10% (v/v) heat-inactivated FBS (56°C for 30 min) in Dulbecco’s PBS pH 7.4.
-
4.
ELISA color detection substrate: reagent A and reagent B (BD OptEIA TMB Substrate Reagent Set; BD Biosciences).
-
5.
Stop solution: 0.2 N H2SO4.
3 Methods
3.1 Tissue Culture
-
1.
Harvest bone marrow aspirates from femurs and tibias of an 8–12 week old C57BL/6 mouse using a 5 ml syringe and a 26 gauge needle to flush the marrow out with IMDM, 10% FCS.
-
2.
Dilute bone marrow aspirates to 40 ml in IMDM, 10% FCS, 150 μM MTG and place cells in a 75 cm2 tissue culture flask to adhere at 37°C, 5% CO2.
-
3.
After 4 h, remove culture supernatant to a 50 ml conical Falcon tube and spin down non-adherent cells (300 × g for 5 min). Count nucleated cells (See Notes 1 and 2).
-
4.
Resuspend cells at 0.5 × 106 cells/ml (i.e., about 160 ml) (See Note 3) in bone marrow macrophage base medium (IMDM, 10% FCS, 150 μM MTG, no additional growth factors).
-
5.
Divide equally into four 75 cm2 filter top tissue culture flasks (about 40 ml per flask), and add 10 ng/ml of recombinant growth factors. Add MCSF to 2 flasks, GM-CSF to 1 flask, and IL-3 to the final flask (See Notes 4 and 5).
-
6.
Replace medium at day 4, spinning down non-adherent cells and returning them to the flask and at day 7, discarding non-adherent cells.
-
7.
At day 7, add IL-4 (10 ng/ml) to 1 of the flasks derived in MCSF alone. Incubate cells for 3 more days (See Notes 6 and 7).
-
8.
At day 10, adherent cells are lifted and replated for stimulations and analyses. Cells are lifted off the tissue culture flask using Cell Dissociation Buffer. Place 5 ml of buffer on cells for 2 min and then bang the side of the flask with the heel of your palm firmly several times. Ensure that cells have lifted off of the flask by examining the flask under the microscope. Remove resuspended cells into a 15 ml conical Falcon tube and wash the flask with an additional 10 ml of IMDM, 10% FBS. Pool and spin down the cells at 300 × g for 5 min. Resuspend cells in a small volume and count viable cells using a hemocytometer.
-
9.
Cells can be harvested into the appropriate buffer for assays (Q-PCR, SDS-PAGE, and Western blotting, or arginase) or replated at a concentration of 0.5 × 106 cells/ml in IMDM, 10% FCS, 150 μM MTG + growth factor used for their derivation (MCSF, GMCSF, IL-3) or treatment (MCSF + IL-4) during growth for stimulations (for NO assays or ELISAs).
-
10.
To stimulate cells, replate in 6 wells (1 ml in a 6-well plate). Add 10 ng/ml LPS to 3 wells and incubate at 37°C, 5% CO2 for 24 h.
-
11.
Harvest cell supernatants to an eppendorf tube and remove contaminating cells by microfuging at 13,000 × g for 5 min. Divide clarified supernatants into two fresh eppendorf tubes and store at −20°C until ready to assay supernatants (See Note 8).
3.2 Quantitative PCR (See Note 9)
-
1.
For RNA isolation, solubilize 105 cells in 100 μl of TRIzol and incubate at 23°C for 5 min (See Note 10).
-
2.
Add 20 μl of chloroform, caps tubes, and shake each sample vigorously by hand and incubate at 23°C for 2 min.
-
3.
Centrifuge at 12,000 × g for 15 min at 4°C and carefully remove upper aqueous phase to a fresh tube (approximately 60 μl).
-
4.
Add 30 μl of isopropanol and incubate at 23°C for 10 min.
-
5.
Centrifuge at 12,000 × g for 15 min at 4°C and remove the isopropanol. Wash one time with 100 μl of 75% ethanol by vortexing and centrifuging at 12,000 × g for 15 min at 4°C. Remove ethanol carefully but thoroughly and allow samples to air-dry at 23°C for 5 min. Do not over dry or dry under vacuum because this will dramatically reduce the solubility of the RNA pellet.
-
6.
Resuspend RNA in 20 μl of DEPC-treated water by gently pipetting up and down. If RNA is difficult to resuspend, heat at 65°C for 5 min and pipet up and down. Quantitate RNA using a NanoDrop Spectrophotometer (See Note 11).
-
7.
For reverse transcription, combine 0.1 μg of RNA for each sample with 1 μl of oligodT and increase volume to 12.5 μl with DEPC-treated H2O. Incubate tubes at 65°C for 5 min and plunge into ice (See Note 12).
-
8.
Prepare a master mix for reverse transcription combining 2.5 μl 10× reaction buffer, 0.625 μl 10 mM (each) dNTPs, 0.5 μl MMLV-RT, and 8.875 μl of DEPC-treated water per reaction. Prepare enough master mix for the number of reactions required +10% extra.
-
9.
Add 12.5 μl of master mix to the side of each reaction tube. Quick-spin to combine contents at the bottom of tube. Incubate at 37°C for 2 min. Incubate at 40°C for 50 min. Incubate at 70°C for 15 min. Store cDNA at −20°C for use in Q-PCR.
-
10.
For quantitative PCR (Q-PCR), dilute cDNA samples 1 in 4 and prepare three serial twofold dilutions for each sample (See Note 13).
-
11.
Prepare a Q-PCR master mix for each primer pair used. For 12.5 ml reactions, combine 0.1875 μl of forward primer (20 μM), 0.1875 μl reverse primer (20 μM), 6.25 μl SYBR green master mix, and 4.875 μl of ddH2O. Prepare enough master mix to do all of the samples in triplicate, a blank sample (no cDNA template) and an additional 10%.
-
12.
Aliquot 11.5 μl of master mix into each well and add 1 μl of template into the reaction mix at the bottom of each well changing tips every time.
-
13.
Cover the plate with the plate sealer (single use transparent film specific to Q-PCR machine).
-
14.
Q-PCR requires melting of the template and activation of the polymerase followed by a two step reaction that alternates between annealing/extension and melting so is programmed as follows: 95°C for 10 min, 40 cycles of: 60°C for 30 s (annealing/extension), followed by 95°C melting for 15 s. Fold differences in gene expression are determined using the software accompanying the light cycler (SDS 2.1) and are compared between samples relative to a housekeeping gene within the sample, as illustrated in Fig. 1.
3.3 SDS-PAGE
-
1.
For SDS-PAGE, 1 × 106 cells can be lysed in 200 μl of 1× LDM. Shear DNA in samples by passing five times through a 26 gauge needle attached to a 1 ml tuberculin syringe (See Note 14). Boil 1 min. Store samples in the freezer at −20°C until ready to load on SDS-PAGE.
-
2.
SDS-PAGE instructions provided here are for preparation of a 40 ml separating gel (1.5 mm thick × 5 cm wide × 16 cm long) to be used with the Bio-Rad Protean II xi Cell. Clean glass plates thoroughly, rinse with water and then rinse with 95% ethanol and air-dry immediately before use. Clean spacers and combs with 95% ethanol, air-dry, and assemble the apparatus as per manufacturers’ instructions (See Note 15).
-
3.
To prepare a 10% separating gel, combine 20 ml distilled water, 10 ml 4× separating gel buffer and 10 ml acrylamide stock solution (wearing gloves) in a 50 ml Falcon tube and mixing by inversion. Degas for 2 min using a vacuum pump or 10 min using a house vacuum (See Note 16).
-
4.
Add APS (80 μL) and 20 μL TEMED and mix gently but thoroughly by rocking the Falcon tube to avoid introducing air bubbles. Pour the entire 40 ml solution between the glass plates. Using a Pasteur pipet, gently add 5 ml of H2O-saturated butanol to overlay the top of the gel. Be careful not to cause mixing with the denser gel solution. Allow gel to polymerize about 30 min.
-
5.
Pour off the alcohol overlay. Rinse the gel top with distilled water and drain water well (See Note 17).
-
6.
To make a 4% stacking gel, combine 9.75 ml water, 3.75 ml 4× stacking gel buffer, and 1.5 ml acrylamide stock solution. Add 75 μl APS and 15 μl TEMED in a 50 ml Falcon tube. Mix by inversion and pipet onto the top of the separating gel. Place the comb into the top of the gel. Avoid trapping air bubbles below or on the side of the comb during insertion. Allow to polymerize for 60 min before removing comb.
-
7.
Using gel loading tips, add 100 μl of the sample (one-half) to bottom of the wells.
-
8.
Prepare 1.4 L of running buffer by diluting 140 ml of 10× running buffer stock solution to 1.4 L with dH2O. Gently add running buffer to top up the wells with a Pasteur pipet and then fill the upper buffer chamber with running buffer. Pour the remaining running buffer into the bottom buffer reservoir of the gel apparatus ensuring that it covers the bottom of the gel and glass plates.
-
9.
Fill the inner chamber of the gel apparatus with cold water and run gel overnight (16 h) at 65 V.
3.4 Western Blotting
-
1.
Instructions provided are for use with the Bio-Rad Trans-Blot Cell. Cut one piece of PVDF membrane and two pieces of Whatman filter paper to 5 cm × 16 cm.
-
2.
Wet PVDF membrane in methanol and the hydrate the membrane by adding 50 ml dH2O. Agitate at room temperature for about 15 min until the water no longer beads or streaks off of the membrane.
-
3.
Disassemble gel apparatus, cut the stacking gel off and discard and soak the separating gel in transfer buffer along with the PVDF membrane.
-
4.
To prepare transfer buffer, combine 400 ml of 10× transfer buffer and 3.2 L dH2O. Finally, add 400 ml methanol (See Note 18).
-
5.
Assemble the gel sandwich on the clear side of the transfer tank holder wetting each piece generously in transfer buffer as you assemble. The gel sandwich is assembled in the following order: 1 Scotch-Brite pad, 1 piece of Whatman filter paper, PVDF membrane, gel (from left to right, note the orientation), 1 piece of filter paper, 1 Scotch-Brite pad. Firmly roll out the gel sandwich with a 10 ml pipet applying downward pressure to thoroughly remove air bubbles trapped between the layers.
-
6.
Secure the gel sandwich in its holding apparatus and move it into the transfer tank. Fill the transfer tank with transfer buffer. Run cold water through the transfer tank constantly during transfer. Transfer gels for 4 h at 0.6 amps. Ensure that the buffer tank does not overheat during transfer. If the transfer apparatus feels too warm, place the entire assembly into a secondary container and pack ice around it.
-
7.
Remove and disassemble the gel sandwich. Peel the membrane back from the gel, and place in a container suitable for probing. Mark the molecular weight markers on the membrane with an indelible pen. Add 50 ml of blocking solution and incubate for 2 h at 23°C on an orbital shaker.
-
8.
Incubate the blocked membrane in primary antibody overnight at 4°C on an orbital shaker (See Notes 19 and 20).
-
9.
Wash the membrane 3 × 10 min in TBST at 23°C on an orbital shaker.
-
10.
Incubate with secondary antibody (anti-mouse-HRP for argI and GAPDH; anti-rabbit-HRP for Ym1) for 45 min at 23°C on an orbital shaker.
-
11.
Wash the membrane 3 × 10 min in TBST at 23°C on an orbital shaker.
-
12.
For ECL detection, combine 7.5 ml of ECL reagent A and 7.5 ml of reagent B together and pipet onto membrane to cover the entire surface. Gently agitate by hand for 1 min.
-
13.
Drain excess fluid from the membrane and place it between two layers of saran wrap and expose to film in a dark room. Exposure times for these antibodies are very short, typically in the range of 5–30 s for Ym1 and GAPDH and 30–60 s for arginase I. Develop film in a film processor (See Note 21). An example of the results produced by this technique is shown in Fig. 2.
3.5 Arginase Assay
-
1.
Lyse 0.25 × 106 macrophages in 50 μL arginase lysis buffer.
-
2.
Determine protein concentration in cell lysates using Bio-Rad protein quantification assay
-
3.
Pipet 5 μg of protein lysate into an eppendorf tube and top up the volume to 100 μL with arginase lysis buffer.
-
4.
Add 10 μL of 10 mM MnCl2 and incubate the samples at 55°C in a water bath for 10 min.
-
5.
Add 100 μL 0.5 M L-arginine into each sample and incubate at 37°C for 1 h (See Note 22).
-
6.
Add 800 μL acid mixture to each sample. Add 40 μL of ISPF solution into each reaction and pipet to mix.
-
7.
To prepare a standard curve, make twofold serial dilutions of urea stock solution in dH2O using dH2O as a blank. Add 100 μl of each to an eppendorf tube. Add 400 μL acid solution and then add 25 μl ISPF to each tube.
-
8.
Boil samples and standards for 30 min in locking eppendorf tubes. Let samples cool to room temperature 23°C in the dark (10 min) (See Note 23).
-
9.
Read absorbance at 550 nm within 30 min. Arginase activity detected ± SD for three independent assays performed in triplicate is shown in Fig. 3a.
3.6 NO Assay
-
1.
To prepare a standard curve, prepare twofold serial dilutions of NaNO2 stock in IMDM, 10% FBS using IMDM, 10% FBS as a blank.
-
2.
Pipet 50 μL of standard, blank or clarified culture supernatant into a flat bottom polystyrene non-tissue-culture-treated 96-well plate.
-
3.
Add 50 μL of sulfanilamide solution into each well.
-
4.
Add 50 μL of naphthylethylenediamine dihyrochloride solution into each well.
-
5.
Incubate the plates for 10 min in the dark. Read the absorbance at 550 nm within 30 min. Nitrite detected ± SD for three independent assays performed in triplicate is shown in Fig. 3b.
3.7 Enzyme-Linked Immunosorbent Assays
-
1.
ELISA kits for IL-12p40 and IL-10 were purchased from BD Biosciences and assays were performed as per manufacturer’s instructions. Cytokine production ± SD from four independent experiments assayed in duplicate are shown in Fig. 4.
4 Notes
-
1.
Nucleated cell counts can be performed by diluting cell suspensions 1 in 20 in 3% acetic acid. This procedure lyses all cells including red blood cells and the remaining nuclei can be counted on a hemocytometer.
-
2.
A thorough bone marrow flush results from two femurs and two tibias gives up to 80 × 106 cells and approximately 90% of cells remain in suspension after 4 h of adherence depletion. The number of cells that become adherent does vary in some genetically modified animals and so this is a very important step if macrophages from genetically modified mice are being compared to wild type mice.
-
3.
It is much easier to resuspend cell pellets in a small volume of medium (5 ml) and then to dilute it up to a larger volume. This ensures a homogeneous cell suspension.
-
4.
We have assayed several different sources of conditioned media and the amount of growth factor that they provide varies dramatically between source and batch. Because the procedure described here aims to compare alternative activation strategies based on growth factors used during differentiation, we recommend that recombinant sources of growth factor are used to obtain similar results.
-
5.
Cell concentration during derivation is important because macrophage skewing during differentiation requires cell intrinsic and cell extrinsic factors. Cell extrinsic factors are affected by cell concentration.
-
6.
Macrophages derived in this way are consistently more than 95% positive for Mac-1 and F4/80 by flow cytometric analysis.
-
7.
Recombinant IL-13 also skews MCSF derived macrophages to an alternatively activate phenotype, although it is less potent than IL-4 when compared directly. IL-10 (10 ng/ml) enhances IL-4 or IL-13 induced alternative activation of macrophages, but it does not mediate skewing macrophages to an M2a phenotype on its own.
-
8.
Cell supernatants harvested for ELISAs should be stored in aliquots because some cytokines are sensitive to freeze–thaw cycles.
-
9.
RNA is extremely sensitive to degradation by ubiquitous RNAses. All sample handling must be done with gloves, all plasticware used should be RNAse free, and all water should be treated with DEPC.
-
10.
TRIzol containing samples should be handled in a fume hood.
-
11.
An A260/A280 ratio of 1.6–2.0 reflects pure and well-solubilized RNA and an A260 of 1.0 abs unit = 40 μg/ml of RNA. Store RNA for up to 6 months at −80°C.
-
12.
Plunging RNA into ice after melting prevents formation of secondary structures that can interfere with reverse transcription.
-
13.
Q-PCR should always be performed in triplicate for each sample and analysis of transcripts is done relative to an unaffected control gene (β-glucuronidase or GUS), which should also be performed in triplicate for each sample.
-
14.
If cell suspensions are too viscous, a larger bore needle can be use to begin to shear the DNA and then decreased until the sample passes easily through a 26 gauge needle.
-
15.
Before pouring your running gel mix into the SDS-PAG apparatus, fill the assembled gel apparatus with dH2O to ensure that it is not leaking.
-
16.
Use a trap between the solution being degassed and the vacuum assembly to avoid contamination with acrylamide, which is a neurotoxin.
-
17.
Do not leave the alcohol on top of the gel for too long, as it can cause the gel to dehydrate.
-
18.
Methanol will cause the salts to precipitate out of the 10× transfer buffer so should be added last to the pre-diluted transfer buffer. However, if this is done in the wrong order, simply add a stir bar and place the slurry onto a stir plate and the precipitate will go back into solution.
-
19.
Antibodies can be “multiplexed” if you are confident that each antibody does not have a cross-reactive band that will affect detection by the other antibodies. Another way to multiplex detection is to cut your membrane horizontally ensuring that you do not cut through a band of interest. For the detection described here, we routinely cut our membrane horizontally between the 55 and 40 kDa molecular weight markers and probe the upper half of the membrane with anti-Ym1 (Mw 55 kDa) and the lower half of the membrane with anti-argI (Mw 36.5 and 38 kDa dimer) and anti-GAPDH (Mw 35 kDa) simultaneously.
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20.
Primary antibodies incubation can be at room temperature for 2 h, but our best experience to minimize background is to incubate overnight (16 h) at 4°C.
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21.
Alternatively activated macrophages are larger than classically activated macrophages so when comparing the same number of macrophages, the protein loading (GAPDH) will increase. For that reason, we routinely harvest enough cells to run our gels twice, the first time comparing equal cell numbers and for a second run, we will adjust our loading according to the loading control to load equal amounts of protein. Our lab does not typically assay for protein prior to loading because some of the additional proteins that we are interested in are extremely sensitive to degradation upon cell lysis and we avoid that problem by resuspending immediately in LDM, shearing and boiling our samples.
-
22.
Increasing this incubation time up to 2 h can increase the sensitivity of this assay if arginase activity is low.
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23.
ISPF will form a precipitate in the reaction mixture. Read absorbance of clear supernatants.
References
Martinez FO, Sica A, Mantovani A, Locati M (2008) Macrophage activation and polarization. Front Biosci 13:453–461
Adamson R (2009) Role of macrophages in normal wound healing: an overview. J Wound Care 18:349–351
Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3:23–35
Gordon S (2007) Macrophage heterogeneity and tissue lipids. J Clin Invest 117:89–93
Nair MG, Gallagher IJ, Taylor MD, Loke P, Coulson PS, Wilson RA, Maizels RM, Allen JE (2005) Chitinase and Fizz family members are a generalized feature of nematode infection with selective upregulation of Ym1 and Fizz1 by antigen-presenting cells. Infect Immun 73:385–394
Munder M (2009) Arginase: an emerging key player in the mammalian immune system. Br J Pharmacol 158:638–651
Yeramian A, Martin L, Arpa L, Bertran J, Soler C, McLeod C, Modolell M, Palacin M, Lloberas J, Celada A (2006) Macrophages require distinct arginine catabolism and transport systems for proliferation and for activation. Eur J Immunol 36:1516–1526
Lee J, Ryu H, Ferrante RJ, Morris SM Jr, Ratan RR (2003) Translational control of inducible nitric oxide synthase expression by arginine can explain the arginine paradox. Proc Natl Acad Sci USA 100:4843–4848
Heinsbroek SE, Gordon S (2009) The role of macrophages in inflammatory bowel diseases. Expert Rev Mol Med 11:e14
Mantovani A, Allavena P, Sica A, Balkwill F (2008) Cancer-related inflammation. Nature 454:436–444
Ho VW, Sly LM (2009) Derivation and characterization of murine alternatively activated (M2) macrophages. Methods Mol Biol 531:173–185
Brombacher F, Arendse B, Peterson R, Holscher A, Holscher C (2009) Analyzing classical and alternative macrophage activation in macrophage/neutrophil-specific IL-4 receptor-alpha-deficient mice. Methods Mol Biol 531:225–252
Rauh MJ, Ho V, Pereira C, Sham A, Sly LM, Lam V, Huxham L, Minchinton AI, Mui A, Krystal G (2005) SHIP represses the generation of alternatively activated macrophages. Immunity 23:361–374
Sinha P, Clements VK, Ostrand-Rosenberg S (2005) Reduction of myeloid-derived suppressor cells and induction of M1 macrophages facilitate the rejection of established metastatic disease. J Immunol 174:636–645
Kuroda E, Ho V, Ruschmann J, Antignano F, Hamilton M, Rauh MJ, Antov A, Flavell RA, Sly LM, Krystal G (2009) SHIP represses the generation of IL-3-induced M2 macrophages by inhibiting IL-4 production from basophils. J Immunol 183:3652–3660
Fleetwood AJ, Lawrence T, Hamilton JA, Cook AD (2007) Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation. J Immunol 178:5245–5252
Kuroda E, Noguchi J, Doi T, Uematsu S, Akira S, Yamashita U (2007) IL-3 is an important differentiation factor for the development of prostaglandin E2-producing macrophages between C57BL/6 and BALB/c mice. Eur J Immunol 37:2185–2195
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Weisser, S.B., McLarren, K.W., Kuroda, E., Sly, L.M. (2013). Generation and Characterization of Murine Alternatively Activated Macrophages. In: Helgason, C., Miller, C. (eds) Basic Cell Culture Protocols. Methods in Molecular Biology, vol 946. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-128-8_14
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