23 Isolation and Differentiation of Murine Macrophages

assay. PCR cycles that generate a linear fit with a slope approximately −3.32 (EPCR = [1–10−(1/slope)] × 100). PCR efficiency between 95 and 105 % are considered acceptable. The linearity is denoted by the Rsq value (correlation coefficient), which should be very close to 1 (>0.985). Another quality indicator of your assay is that there should be a difference of approximately 3.3 in CT values between two standard curve points with a tenfold dilution. 23. Most PCR products will melt somewhere in the range of 80–90 °C, although this melting point can vary with the size and sequence of your specific target. Ideally, the experimental samples should yield a single sharp peak within this temperature range, and the melting temperature should be the same for all the samples. Furthermore, both water and −RT should not generate significant fluorescent signal. If the dissociation curve reveals a series of peaks, it indicates that there is not enough discrimination between specific and nonspecific reaction products (for example, due to dimerization of primers), which would render optimization of the qRT-PCR necessary.

Acknowledgement

A.M.B. is supported through the Ramón y Cajal program (RYC-2010-06473). A.N.D.C. was supported by CIHR and by University of Glasgow.

References

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PS, Okine EK, Dodson MV (2004) Primary adipocyte culture: adipocyte purification methods may lead to a new understanding of adipose tissue growth and development.

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Am J Physiol Cell Physiol 302:C327–C359 13. Fleige S, Pfaffl MW (2006) RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med 27:126–139 14. Thellin O, Zorzi W, Lakaye B, De Borman B,

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RT-PCR. Physiol Genomics 12:163–174

Chapter 23

Isolation and Differentiation of Murine Macrophages

Francisco J. Rios, Rhian M. Touyz, and Augusto C. Montezano

Abstract

Macrophages play a major role in inflammation, wound healing, and tissue repair. Infiltrated monocytes differentiate into different macrophage subtypes with protective or pathogenic activities in vascular lesions. In the heart and vascular tissues, pathological activation promotes cardiovascular inflammation and remodeling and there is increasing evidence that macrophages play important mechanisms in this environment. Primary murine macrophages can be obtained from: bone marrow by different treatments (granulocytemacrophage colony-stimulating factor—GM-CSF, macrophage colony-stimulating factor—M-CSF or supernatant of murine fibroblast L929), peritoneal cavity (resident or thioglycolate elicit macrophages), from the lung (alveolar macrophages) or from adipose tissue. In this chapter we describe some protocols to obtain primary murine macrophages and how to identify a pure macrophage population or activation phenotypes using different markers.

Key words Macrophage, Murine, Peritoneum, Bone marrow, Resident, Thioglycollate, L929 supernatant

1 Introduction

Macrophages play a central role in inflammatory response and host defense, contributing the establishment of the innate immunity. Additionally these cells participate in homeostatic functions, including fibrosis, lipid metabolism, and tissue remodeling [1].

The most known process involved in macrophage activation is the phagocytosis. Monocytes from the blood vessels migrate to the tissue, where they encounter and engulf microorganisms. This process is followed by production and secretion of several mediators that increase the inflammatory response, such as tumor necrosis factor-α (TNF-α) and nitric oxide. On the other hand, in some situations, macrophages participate in tissue remodeling, secreting anti-inflammatory substances, such as interleucin-10 (IL-10) and transforming growth factor beta (TGFβ), that will control the inflammatory response, contributes to collagen production and tissue remodeling [2].

Rhian M. Touyz and Ernesto L. Schiffrin (eds.), Hypertension: Methods and Protocols, Methods in Molecular Biology, vol. 1527, DOI 10.1007/978-1-4939-6625-7_23, © Springer Science+Business Media LLC 2017 297

Macrophages have an extraordinary plasticity to change their phenotype according to the receptors that have been activated and the microenvironment where they are located. These cells can be polarized to classical activated phenotype (M1 macrophages), involved in the pro-inflammatory response or to an alternative activated phenotype (M2 macrophages) that are mostly involved in anti-inflammatory mechanism, fibrosis, and wound healing. M1 macrophages have high expression of Fc receptors and produce pro-inflammatory cytokines (MCP-1, IL-6, TNF, IL-12) and nitric oxide. Inversely, M2 macrophages have high expression of mannose receptor, arginase-1 and produce IL-10 and TGF-β. Although some studies have found the presence of both phenotypes in cardiovascular diseases, the main role of M1 and M2 macrophages in this context remains unclear [1, 2].

Macrophages in the tissue produce reactive oxygen species and metalloproteinases that will increase the inflammatory response and activate smooth muscle and endothelial cells, contributing to the cardiovascular disease [3]. In fact, it has been show that the activation of mineralocorticoid receptor in macrophages is related to oxidative stress and inflammation, playing a role in cardiac hypertrophy and blood pressure [4].

In this chapter we describe protocols to obtain murine macrophage in vitro. Depending on the method used, it can be obtained from different macrophage populations, which have different properties in the inflammatory response.

2 Murine Macrophage Identification

Murine macrophages can be identified by expressing F4/80 and CD11b (Mac1), MOMA-1 [5]. The antibodies required for these markers are supplied by different companies, they are well acceptable among the scientists and can be used for flow cytometry, immunohistochemistry, and immunofluorescence.

3 Culture of Murine Bone Marrow Derived Macrophages

This is a saving cost method to obtain macrophage population from murine bone marrow (Fig. 1). Macrophages can be differentiated from the bone marrow by treatment with murine recombinant M-CSF (10 ng/mL). However, several studies have demonstrated that L929 supernatant promotes differentiation of macrophages by producing M-CSF [6]. This is well accepted as a pure macrophage population model. It has been described that bone marrow derived macrophages are an important model to study reactive oxygen species production and endothelial cell adhesion [7].

3.2 Methodology

– Phosphate buffered saline (PBS), pH 7.2 – 10-mL syringe. – 26 G × 1/2″ needle (BD Becton, Dickinson and Company). – Forceps and scissors. – Centrifuge at 4 °C with rotor for 50 mL tube. – 100 × 15-mm sterile bacterial plastic petri dishes. – 50-mL polypropylene tubes. – Cell strainers, 70 μm (BD Falcon™). – 70 % ethanol in water. – L-Cell Conditioned Medium (LCM): DMEM (prepared as in item 3.1), 20 % of L929 cell culture supernatant, 15 % of FCS).

Supernatant of the murine fibroblast L929 is obtained by growing 1 × 105 cells/mL in DMEM (fresh medium prepared according to item 3.1, supplemented with 10 % FCS, 50 μM 2-Mercaptoethanol). Usually we add 60 mL of the cell suspension in a 150 cm2 flask. Let the cells grow in a humidified incubator 5 % CO2 at 37 °C for 7 days, to accumulate growth factor in the supernatant. At day 7, remove the supernatant, wash the cells monolayer with PBS and harvest them using cell scraper. Centrifuge the supernatant at 5000 × g, 10 min, filter at 0.22 μm and freeze at −80 °C (Note 2). 1. Sacrifice the mouse according to the ethical permission from your research institute. 2. Rub a piece of cotton embedded in alcohol along the abdomen of the mouse. Make an incision in the abdomen and with scissor remove the muscles from the bones to be able to expose the femur head. Be careful you do not break the femur. Soak the cleaned bone in ice-cold PBS until the extraction of the other bones has finished. Until this step the work can be performed in normal laboratory bench. 3. From this step the work must be done in a laminar flow hood.

Sterilize the bones by soaking in alcohol for 10 s and keep them in ice-cold sterile PBS. 4. Carefully take the bone with a forceps and with a scissor, cut a small piece of both extremities. Using a 26 G × 1/2″ needle in a 10 mL syringe, flush the bones with cold PBS. Repeat the flush until the bone change the color to white. 5. With a 10 mL pipette, do movement up and downs to be able to dissolve the cells aggregates. 6. Pass the cell suspension through a cell strainer (70 μm) to get a single cell suspension. After, wash the cell strainer with 5 mL of cold PBS. 7. Centrifuge the cell suspension at 400 × g for 10 min.

8. Resuspend the cells in 10 mL of L-Cell conditioned Medium, count and adjust the cell concentration to 2 × 106 cells/mL. 9. Plate 10 mL of the cell suspension in a 100 × 15-mm sterile bacterial plastic Petri dishes. Incubate the plates in a humidified incubator with 5 % CO2 at 37 °C. At day 3, add 15 mL of the L-Cell conditioned Medium. 10. At day 6, the cells are already differentiated in bone marrow derived macrophages and are strongly attached to the plastic (Note 1). 11. To detach the cells. Remove the LCM and add 10 mL of cold

PBS. Place the plates with cells and PBS on ice for 30 min. By using a cell scraper, gently scrape the cells off the plate. 12. Centrifuge the cells at 300 × g for 5 min. Resuspend in

DMEM/5 % FCS and plate according to the protocol required for the experiment. Stimulate the cells after 24 h (Note 3–4).

4 Culture of Resident Peritoneal Macrophages

4.1 Materials

– RPMI1640 (containing l-glutamine, 20 mM HEPES, and penicillin and streptomycin). – Endotoxin-free heat-inactivated fetal calf serum (FCS). – Phosphate buffered saline (PBS), pH 7.2. – 10-mL syringe. – 26 G × 1/2″ and 21 G needles (BD Becton, Dickinson and

Company). – Forceps and scissors. – Centrifuge at 4 °C with rotor for 50 mL tube. – 50-mL polypropylene tubes. – 70 % ethanol in water.

1. Sacrifice the mice according to the ethical permission from your research institute. 2. Sterilize the mouse, by placing it in 70 % ethanol for 5 s. 3. Using a forceps, delicately pitch up the skin of the inferior ventral abdomen and make an incision with a scissors to expose all peritoneum. Be careful you do not injure the peritoneal membrane. 4. Pull up the peritoneal membrane using a forceps and with 26 G × 1/2″ inject 6 mL of ice-cold PBS in the peritoneal cavity. Do not pierce the visceral organs or will contaminate the macrophages.

4.2 Methodology (Fig. 2)

Fig. 2 Extraction of resident peritoneal macrophages. This is a simplified flowchart for the isolation of resident peritoneal macrophages. PBS phosphate buffered saline, RPMI Roswell Park Memorial Institute medium

5. Shake the mouse for 10 s. This step will detach macrophage from the peritoneal cavity and get a cell suspension. 6. Using the 21 G needle in a 10 mL syringe, remove the macrophages suspension from the peritoneal cavity. Insert the needle with beveled end facing down in the opposite side of the liver and distant from the fatty tissue. The needle might gets stack with the fat, to solve this problem removed the needle and insert it again.

7. Collect the cell suspension in a 50 mL tube containing 20 mL of ice-cold PBS and placed on ice. 8. Centrifuge the cells suspension at 350 × g at 4 °C for 5 min. 9. Resuspend the cell pellet with RPMI 1640 supplemented with 5 % of FCS. Count the cells, plate then at 2 × 106 cells/mL and incubate for 2 h at 37 °C. 10. Gently, wash the non-adherent cells with PBS at 37 °C. Wash gently because resident peritoneal macrophages do not attach strongly. 11. Add RPMI 1640 with 5 % FCS to the adherent macrophages and proceed with your experiments. Usually with this protocol you might obtain 4–6 × 106 peritoneal cells and approximately 50 % are macrophages.

5 Thioglycollate Elicited Macrophages

5.1 Materials

5.2 Methodology

Thioglycollate medium is often used to increase the amount of macrophages recruited to the peritoneal cavity. This procedure can be used to obtain activated inflammatory macrophages from mice, or it can be used as an analytical procedure to compare rates of leukocyte accumulation by different treatments. This protocol might results approximately 2×107 of inflammatory macrophages.

– RPMI1640 (containing l-glutamine, 20 mM HEPES, and penicillin and streptomycin). – Endotoxin-free heat-inactivated fetal calf serum (FCS). – Phosphate buffered saline (PBS), pH 7.2. – 10-mL syringe. – 26 G × 1/2″ and 21 G needles (BD Becton, Dickinson and

Company). – Forceps and scissors. – Centrifuge at 4 °C with rotor for 50 mL tube. – 50 mL polypropylene tubes. – 70 % ethanol in water. – 3 % brewer modified thioglycollate medium (3 % w/v of an autoclaved stock prepared from dehydrated thioglycollate medium and sterile water).

1. Using a 26 G × 1/2″ needle, inject 1 mL of 3 % thioglycollate medium into the peritoneal cavity of the mouse.

2. After 4 days, thioglycollate elicited macrophages might be obtained from the peritoneal cavity (follow the protocol in

Subheading 4.2, steps 1–8). 3. Plate the peritoneal cells in a concentration 1 × 106 cells/mL and incubate for 2 h at 37 °C. 4. Wash the non-adherent cells with PBS at 37 °C. Thioglycolate elicited macrophages attach strongly to the plate. 5. Add RPMI 1640 with 5 % FCS to the adherent macrophages and proceed with your experiments.

6 Alveolar Macrophages

6.1 Materials

6.2 Methodology

Typically, a total of ~3–5 × 105 alveolar macrophages cells can be obtained per mouse. Therefore this procedure is often performed on groups of ten mice or more to provide sufficient alveolar macrophages for functional assays (Fig. 3).

– RPMI 1640 (containing l-glutamine, 20 mM HEPES, and penicillin and streptomycin). – Endotoxin-free heat-inactivated fetal calf serum (FCS). – Anesthetics: Cocktail of ketamine-HCl 100 mg/kg and xylazine-HCl 10 mg/kg in PBS. – PBS containing 0.5 mM EDTA and without calcium and magnesium. – Forceps and scissors. – 18 G needles and 10-mL syringes. – 50-mL polypropylene tubes.

1. Anesthetize mice by injecting 100–200 μL of the Anesthetics

Cocktail ketamine–xylazine cocktail. Do not sacrifice the mouse by cervical dislocation, because it might injure and contaminate the cells with blood. Also, do not use CO2 asphyxia, because it might change the alveolar macrophage response. 2. In a laminar flow hood. Rub a piece of cotton embedded in alcohol along the neck and abdomen of the mouse. Make a small incision in the neck and expose the trachea on the ventral side. Insert a18 G needle in the trachea (just below the larynx) and tie up with a threat. 3. Hold a 5-mL syringe in the needle and slowly inject sterile 2–3 mL of PBS/EDTA at 37 °C under gentle massage of the lungs. 4. Gently pull the plunger of the syringe back to withdraw the cell suspension and collect it in a 50 mL tube containing

7. Plate the cells and incubate in a humidified incubator with 5 %

CO2 at 37 °C for 2 h. 8. Gently, wash the non-adherent cells with PBS at 37 °C. 9. Add RPMI 1640 with 5 % FCS to the adherent macrophages and proceed with your experiments.

7 Isolation of Macrophages from Adipose Tissue

7.1 Materials

7.2 Methodology (Fig. 4)

– RPMI1640 (containing l-glutamine, 20 mM HEPES, and penicillin and streptomycin). – Endotoxin-free heat-inactivated fetal calf serum (FCS). – PBS, pH 7.2 at room temperature. – Forceps and scissors. – 15 and 50-mL polypropylene tubes. – Collagenase type 1 (1 mg/mL in PBS). – Water bath at 37 °C. – P1000 pipette. – CD11b MicroBeads for mouse samples (MACS Cell Separation

System; Miltenyi Biotec).

1. Sacrifice the mice according to the ethical permission from your research institute. 2. Rub a piece of cotton embedded in alcohol along the abdomen of the mouse. Make an incision in the abdomen and in the peritoneal cavity to expose the abdominal fatty tissue. 3. With forceps and scissor remove the fatty tissue from the abdominal cavity and place it in a 15 mL tube containing collagenase. Digest for 30 min in a water bath at 37 °C and in constant agitation. If you have to extract the adipose tissue from several mice, the fatty can be collected in PBS until the extraction of the samples have finished. 4. Homogenize the digested samples with P1000 pipette by up and down movements. 5. Transfer the digested sample to a 50 mL tube and immediately add 30 mL of cold RPMI 1640 supplemented with FCS 10 % to inactivate the collagenase. 6. Centrifuge the samples at 350 × g at 4 °C for 10 min.

Macrophages will be on the cell pellet and the floating fraction is enriched in adipocytes. 7. Resuspend the cell pellet in 5 mL of cold PBS and filter through the cell strainer. After, wash the cell strainer with 5 mL of cold

PBS.

Table 1 Murine macrophage phenotype markers

M1 M2 References

FIZZ1 ↓ ↑ [8] Mannose receptor (CD206) ↓ ↑ [9] Arginase 1 ↓ ↑ [10] Ym1 ↓ ↑ [8]

Galectin 3 ↓ ↑ [1]

IL-10 ↓ ↑ [2] TNFα ↑ ↓ [12] iNOS ↑ ↓ [13]

IL-12 ↑ ↓ [12]

IL-6 ↑ ↓ [14] IL-1β ↑ ↓ [14]

8 Macrophage Markers

M1 and M2 macrophages can be obtained in vitro by treatment with different cytokine combination. After macrophage differentiation according to item 3, M1 macrophages are obtained by treatment with LPS 50 ng/mL and 5 U/mL of interferon-γ for 24. M2 macrophages are obtained by treatment with IL-4 (20 ng/mL) for 24 h.

M1 and M2 macrophages might be identified according to Table 1:

9 Notes

1. Trypsin can be used instead of cell scraper; however, in our experience, cells stand more passages by using cell scraper. Save the supernatant at −80 °C. Try to test each batch of supernatant and after collect the supernatants in the same flask for the macrophages differentiate with the same concentration of growth factors. Constantly test the L929 cell for mycoplasma contamination. 2. Differentiated macrophages attach strongly to the tissue culture. Common sterile plastic Petri dishes will allow the macrophages to detacheasier and get high number of viable cells than the plates treated for cell culture.