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Immunology and Infection

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Published: June 23rd, 2023

DOI:

10.3791/64566

1Department of General Pathology, Institute of Biological Sciences, Federal University of Minas Gerais, 2Department of Cell Biology and Molecular Genetics, University of Maryland

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-γ and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

Monocytes and macrophages are mononuclear phagocytes that can be derived from progenitors in the bone marrow. Recent studies have reported that macrophages also originate from yolk sac-derived erythro-myeloid progenitors1. Regardless of their derivation, these leukocytes have important effector functions in homeostasis and inflammation2,3. Monocytes are cells from peripheral blood that can further differentiate into macrophages in the tissue2,4, whereas macrophages are heterogeneous cells that exhibit phenotypes and functions re....

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This protocol was carried out in accordance with The National Council for the Control of Animal Experimentation (Concea) and with the approval of the Ethics and Use of Animals Committee (CEUA). C57BL/6 mice were purchased from Biotério Central of Federal University of Minas Gerais (UFMG), Belo Horizonte, Brazil. Personal protective equipment (PPE) such as laboratory coats, gloves, and eye protection should be used at all steps described in this protocol.

1. Preparation of fibroblast.......

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Macrophages are large and adherent cells with special physiological characteristics. They show a diversity of morphologic presentations in culture because of the ability to adhere to glass and plastic, and their typical spread-out morphology is related to the emission of cytoplasmic extensions (Figure 1). Once bone marrow progenitors are exposed to M-CSF from L-929 cell supernatant and start the transformation to mature macrophages, they become adherent to the Petri plate.

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Producing a population of bone marrow-derived macrophages is a basic step in many cell biology experimental models, especially when it is important to achieve a homogeneous population of primary cells. As mentioned, cell progenitors can only transform into macrophages in the presence of M-CSF. L-929 cell supernatants can be used as the main source of M-CSF. Other than the cost, there is no problem using recombinant M-CSF itself8,17. There is some evidence that re.......

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This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), through Rede de Pesquisa em Doenças Infecciosas Humanas e Animais do Estado de Minas Gerais (RED-00313-16) and Rede Mineira de Engenharia de Tecidos e Terapia Celular - REMETTEC (RED-00570-16), and the Brazilian National Council for Scientific and Technological Development (CNPq).

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NameCompanyCatalog NumberComments
20-cc syringeDESCARPACKSI100S4GSterile syringe
26-G needlesBD497AQDKT7Sterile needles
50-ml conical centrifuge tubeSARSTEDT62547254Plastic conical tubes suitable for centrifugation
70% ethanol solutionEMFAL490Ethanol solution for esterilization
Anatomical dissection forceps3B SCIENTIFICW1670Maintain in sterile beaker containing 70% ethanol solution
C57Bl/6 wild type mousePurchased from Biotério Central at Federal University of Minas GeraisNot applicableMice must be specific-pathogen-free, age between 6 and 10 weeks. Mice need be accommodated at least one week earlier for recovering from the stress of transportation
Cell culture flask T175GREINERC7481-50EAT-175 flask, canted neck, surface area 75 cm2, with filter cap, DNase free, RNase free
Cell culture flask T75GREINERC7231-120EAT-75 flask, canted neck, surface area 75 cm2, with filter cap, DNase free, RNase free
Disinfecting or baby Wipes₁CLOROXNot applicableIt helps cleaning the bone
Distilled WaterGIBCO15230Sterile distilled water
DMEM/F12-10Not applicableNot applicableAdd 10 mL of Fetal Bovine Serum (FBS) and 1 mL of Penicillin/ Streptomycin (P/S) to DMEM/F12 (q.s.p. 100 mL)
DMEM/F12-10 + supernatant of L-929 cellsNot applicableNot applicableAdd 20% of supernatant of L929 cells culture on DMEM/F-12-10
Dulbecco′s Modified Eagle′s Medium - F12 (DMEM/F12)GIBCO12500096DMEM: F-12 Medium contains 2.5 mM L-glutamine, 15 mM HEPES, 0.5 mM sodium pyruvate, and 1200 mg/L sodium bicarbonate.Resuspend powder to 1 liter of
distilled water and add 3,7 g of sodium bicarbonate. Adjust pH to 7,2 and filter with 0,22 µM. Storage at 2 - 8 °C freezer
Fetal Bovine Serum, certified, heat inactivated, United States (FBS)GIBCO10082147Enrichment for DMEM-F12
HemocytometerSIGMA-ALDRICHZ359629Used to count macrophages at microscopy
L-929 cellsSIGMA-ALDRICHATCC # CCL-1L-929 is a lineage of mouse fibroblast cells used as a source of macrophage colony stimulating factor (M-CSF)
Nikon TI eclipse NIKON Not applicableNikon TI Eclipse is a fluorescence and phase contrast microscope
Non-enzymatic cell dissociation solutionCELLSTRIPER (CORNING)25-056-CINon-enzimatic cell dissociation solution remove macrophages from the plate without damaging them
Non-treated round culture dishes 100 × 20–mmCORNINGCLS430591Do not use tissue culture treated petri dishes or any tissue culture treated plate
P3199 Penicillin G Potassium SaltUSBIOLOGICAL113-98-4Antibiotics
Penicillin and streptomycin (P/S) solutionUse 0,26 grams of penicillin and 0,40 grams of streptomycin. Mix with 40 mL of sterile PBS. Inside a horizontal laminar flow cabinet, use a 0,22 µM filter and store aliquots of 1.0 mL in 1.5 ml microfuge tubes in the freezer (-20°C)
Phosphate Buffered Saline free from Calcium and Magnesium (PBS)MEDIATECH21-040-CMSterile
S7975 Streptomycin Sulfate, 650-850U/mgUSBIOLOGICAL3810-74-0Antibiotics
Serological pipettes of 10mL or 25 mLSARSTEDT861254001Serological pipettes is used volumes higher than 1 mL
Sodium BicarbonateSIGMA-ALDRICH144-55-8pH correction for DMEM-F12
Software Nis Elements ViewerNIKONNot applicableNIS-Elements Viewer is a free standalone program to view image files and datasets
Sterile PBS and 2% of P/S solutionLABORCRIN590338Add 1 mL of P/S in 40 mL of sterile PBS
Straight iris scissorsKATENANot applicableMaintain in sterile beaker containing 70% ethanol solution
Supernatant of L-929 cellsNot applicableNot applicableL-929 is a lineage of mouse fibroblast cells used as a source of macrophage colony stimulating factor (M-CSF). L-929 supernatant was obtain from the protocol
Surgical Scalpel Blade No.24 Stainless SteelSWANN-MORTON311Used for exposing epiphysis from bones
Trypan Blue Solution, 0.4%GIBCO15250061Trypan Blue Solution, 0.4%, is routinely used as a cell stain to assess cell viability using the dye exclusion test
Trypsin/EDTA solution 0,05%GIBCO25300-062Used to dissociate cells from culture bottle
Water for Injection (WFI) for Cell CultureGIBCOA12873Sterile and endotoxin-free water

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