Chapter 14

Generation and Characterization of Murine Alternatively

Activated Macrophages
Shelley B. Weisser, Keith W. McLarren, Etsushi Kuroda,
and Laura M. Sly
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 IFNg 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-infl ammatory 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: Macrophage phenotype, Macrophage polarization, Alternative activation, Bone marrow
derived macrophages, Macrophage colony stimulating factor, Granulocyte macrophage colony stimulating factor, IL-3, IL-4, Inducible nitric oxide synthesis, Nitric oxide, Arginase I, IL-12p40, IL-10

Cheryl D. Helgason and Cindy L. Miller (eds.), Basic Cell Culture Protocols, Methods in Molecular Biology, vol. 946,
DOI 10.1007/978-1-62703-128-8_14, Springer Science+Business Media, LLC 2013

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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 IFNg or LPS and produce robust amounts of reactive oxygen species and pro-inflammatory cytokines (IL-12, TNFa,
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 RELMa) (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

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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 (1214). 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 (1517).

2. Materials
2.1. Tissue Culture

1. C57BL/6 mouse (812 weeks old). Animals housed and

sacrificed according to institutional requirements.
2. Iscoves Modified Dulbeccos Medium (IMDM).
3. Fetal bovine serum.
4. Recombinant murine macrophage colony stimulating factor
(MCSF or CSF-1).
5. Recombinant murine granulocyte-macrophage colony stimulating factor (GM-CSF).
6. Recombinant murine interleukin-3 (IL-3).
7. Recombinant murine interleukin-4 (IL-4).
8. Monothioglycerol (MTG).

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9. Culture medium: IMDM, 10% FCS, 150 mM MTG. Combine

and filter-sterilize using low protein binding 0.2 micron filter.
Store at 4C for up to 1 month.
10. Cell Dissociation Buffer (Gibco-BRL, Bethesda, MA).
11. Lipopolysaccharide from Escherichia coli serotype O127:B8 (LPS).
12. Incubator set at 37C and 5% CO2.
2.2. Quantitative PCR

1. RNAse-free 1.5 ml eppendorf tubes.

2. Sterile and RNAse-free filter tips.
3. TRIzol (Invitrogen).
4. Isopropanol.
5. 75% (v/v) ethanol in diethylpyrocarbonate (DEPC)-treated
water.
6. Oligo p(dT)20-40 (5 U per 750 ml of DEPC-treated water).
7. dNTPs mix (10 mM 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

1. Laemmlis digestion mix (LDM): 75 mM TrisHCl pH 6.8,

7.5% (w/v) glycerol, 200 mM b-mercaptoethanol, 1.5% (w/v)
bromophenol blue.
2. Separating gel mix (4): 1.5 M TrisCl pH 8.8, 0.4% SDS.
3. Stacking gel mix (4): 0.3 M TrisHCl pH 6.8, 0.4% SDS.
4. 40% Acrylamide/bisacrylamide solution (37.5:1 with 2.6% C).
5. N,N,N,N-Tetramethyl-ethylenediamine (TEMED).
6. 10% (w/v) Ammonium persulfate (APS). Freeze aliquots at
20C for up to 3 months, thawing an aliquot for use and storing at 4C for no more than 7 days.
7. PageRuler prestained protein ladder (Fermentas Life Sciences,
Thermofisher).

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8. Running buffer (10): 0.25 M Trizma base, 1.9 M glycine, 1%

(w/v) sodium dodecyl sulfate (SDS).
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

1. Transfer buffer (10): 0.25 M Trizma base, 1.92 M glycine,

0.5% (w/v) SDS.
2. Methanol.
3. Immobilon-P membrane (0.45 mm pore polyvinyldifluoride
(PVDF)) (Bio-Rad Laboratories, Hercules, CA).
4. Whatman filter paper.
5. Tris-buffered saline with Tween-20 (TBST): 137 mM NaCl,
2.7 mM KCl, 25 mM TrisCl 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
TrisCl 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) a-isonitrosopropiophenone
(aISPP) in absolute ethanol.
8. Bio-Rad protein quantification assay (Bio-Rad, Hercules,
CA, USA).

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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 (C 10H 7NHCH 2
CH2NH2-2HCl-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 (56C for
30 min) in Dulbeccos 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

812 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 mM MTG and place cells in a 75 cm2 tissue culture flask to
adhere at 37C, 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 mM 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 nonadherent 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

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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 mM 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 37C, 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 20C until ready to assay supernatants (see Note 8).
3.2. Quantitative PCR
(see Note 9)

1. For RNA isolation, solubilize 105 cells in 100 ml of TRIzol and

incubate at 23C for 5 min (see Note 10).
2. Add 20 ml of chloroform, caps tubes, and shake each sample
vigorously by hand and incubate at 23C for 2 min.
3. Centrifuge at 12,000 g for 15 min at 4C and carefully remove
upper aqueous phase to a fresh tube (approximately 60 ml).
4. Add 30 ml of isopropanol and incubate at 23C for 10 min.
5. Centrifuge at 12,000 g for 15 min at 4C and remove the
isopropanol. Wash one time with 100 ml of 75% ethanol by
vortexing and centrifuging at 12,000 g for 15 min at 4C.
Remove ethanol carefully but thoroughly and allow samples to
air-dry at 23C 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 ml of DEPC-treated water by gently
pipetting up and down. If RNA is difficult to resuspend, heat
at 65C for 5 min and pipet up and down. Quantitate RNA
using a NanoDrop Spectrophotometer (see Note 11).
7. For reverse transcription, combine 0.1 mg of RNA for each
sample with 1 ml of oligodT and increase volume to 12.5 ml
with DEPC-treated H2O. Incubate tubes at 65C for 5 min
and plunge into ice (see Note 12).
8. Prepare a master mix for reverse transcription combining 2.5 ml
10 reaction buffer, 0.625 ml 10 mM (each) dNTPs, 0.5 ml
MMLV-RT, and 8.875 ml of DEPC-treated water per reaction.

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Prepare enough master mix for the number of reactions

required +10% extra.
9. Add 12.5 ml of master mix to the side of each reaction tube.
Quick-spin to combine contents at the bottom of tube. Incubate
at 37C for 2 min. Incubate at 40C for 50 min. Incubate at
70C for 15 min. Store cDNA at 20C 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 ml of forward primer
(20 mM), 0.1875 ml reverse primer (20 mM), 6.25 ml SYBR
green master mix, and 4.875 ml 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 ml of master mix into each well and add 1 ml 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: 95C for 10 min, 40 cycles of: 60C for 30 s (annealing/extension), followed by 95C 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 ml 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 20C 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).

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Fig. 1. Q-PCR analysis of gene expression of argI and iNOS. Bone marrow aspirates were differentiated into macrophages
in the presence of 10 ng/ml of MCSF, GMCSF, or IL-3 for 10 days or in the presence of MCSF for 7 days followed by addition
of 10 ng/ml of IL-4 for an additional 3 days. After 10 days, macrophages were treated with 10 ng/ml of LPS for 24 h and
cells were harvested into TRIzol for RNA extraction. cDNA was prepared by reverse transcription and used for Q-PCR analysis. Expression of argI and iNOS were evaluated for each sample relative to b-glucuronidase (GUS) as an internal control.
Data shown are means SD for three independent experiments performed in triplicate.

4. Add APS (80 mL) and 20 mL 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 ml APS and 15 ml 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 ml 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

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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 23C on an orbital shaker.
8. Incubate the blocked membrane in primary antibody overnight at 4C on an orbital shaker (see Notes 19 and 20).
9. Wash the membrane 3 10 min in TBST at 23C 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 23C
on an orbital shaker.
11. Wash the membrane 3 10 min in TBST at 23C 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 530 s for Ym1 and GAPDH and 3060 s for arginase I.

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IL-3

235

IL-4
Ym1
ArgI
GAPDH

Fig. 2. Western blot analysis of M2 macrophage marker protein expression. Bone marrow
aspirates were differentiated into macrophages in the presence of 10 ng/ml of MCSF,
GMCSF, or IL-3 for 10 days or in the presence of MCSF for 7 days followed by addition of
10 ng/ml of IL-4 for an additional 3 days. Macrophage cell lysates were separated on a
10% SDS-PAGE, transferred onto PVDF and probed for M2 macrophage markers, Ym1 and
argI, as well as GAPDH, as a loading control.

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 mL arginase lysis buffer.

2. Determine protein concentration in cell lysates using Bio-Rad
protein quantification assay
3. Pipet 5 mg of protein lysate into an eppendorf tube and top up
the volume to 100 mL with arginase lysis buffer.
4. Add 10 mL of 10 mM MnCl2 and incubate the samples at 55C
in a water bath for 10 min.
5. Add 100 mL 0.5 M L-arginine into each sample and incubate
at 37C for 1 h (see Note 22).
6. Add 800 mL acid mixture to each sample. Add 40 mL 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 ml of each to an eppendorf tube. Add 400 mL acid solution
and then add 25 ml ISPF to each tube.
8. Boil samples and standards for 30 min in locking eppendorf
tubes. Let samples cool to room temperature 23C 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 mL of standard, blank or clarified culture supernatant into a flat bottom polystyrene non-tissue-culture-treated
96-well plate.

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3. Add 50 mL of sulfanilamide solution into each well.

4. Add 50 mL 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 manufacturers instructions. Cytokine production SD from four
independent experiments assayed in duplicate are shown in
Fig. 4.

Fig. 3. Analysis of enzymatic activity of argI and iNOS. Bone marrow aspirates were differentiated into macrophages in the
presence of 10 ng/ml of MCSF, GMCSF, or IL-3 for 10 days or in the presence of MCSF for 7 days followed by addition of
10 ng/ml of IL-4 for an additional 3 days. After 10 days, macrophages were harvested into arginase lysis buffer for arginase
activity assays or left untreated or treated with 10 ng/ml of LPS for 24 h. After LPS treatment, cell supernatants were
harvested, clarified and subjected to the Griess assay to measure NO2, downstream of NO production. Data shown are
means SD for three independent experiments performed in triplicate.

Fig. 4. Measurement of pro-inflammatory (IL-12 p40) and anti-inflammatory (IL-10) cytokine production and IL-12/IL-10
ratios. Bone marrow aspirates were differentiated into macrophages in the presence of 10 ng/ml of MCSF, GMCSF, or IL-3
for 10 days or in the presence of MCSF for 7 days followed by addition of 10 ng/ml of IL-4 for an additional 3 days.
Resulting macrophages were treated with LPS for 24 h and cell supernatants were harvested, clarified and assayed by
ELISA for IL-12p40 and IL-10 and the ratio of IL-12/IL-10 produced in response to LPS was calculated. Data shown are
means SD for four independent experiments assayed in duplicate.

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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 freezethaw 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.

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S.B. Weisser et al.

11. An A260/A280 ratio of 1.62.0 reflects pure and well-solubilized

RNA and an A260 of 1.0 abs unit = 40 mg/ml of RNA. Store
RNA for up to 6 months at 80C.
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 (b-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.
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 4C.
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

14

Murine Alternatively Activated Macrophages

239

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.
23. ISPF will form a precipitate in the reaction mixture. Read
absorbance of clear supernatants.
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