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. 2019 Jul 20;9(14):e3302.
doi: 10.21769/BioProtoc.3302.

Isolation and Long-term Cultivation of Mouse Alveolar Macrophages

Clara Jana-Lui Busch  1   2 , Jérémy Favret  1   2   3 , Laufey Geirsdóttir  3 , Kaaweh Molawi  2   3 , Michael H Sieweke  1   2   3
Affiliations

Isolation and Long-term Cultivation of Mouse Alveolar Macrophages

Clara Jana-Lui Busch et al. Bio Protoc. .

Abstract

Alveolar macrophages (AM) are tissue-resident macrophages that colonize the lung around birth and can self-maintain long-term in an adult organism without contribution of monocytes. AM are located in the pulmonary alveoli and can be harvested by washing the lungs using the method of bronchoalveolar lavage (BAL). Here, we compared different conditions of BAL to obtain high yields of murine AM for in vitro culture and expansion of AM. In addition, we describe specific culture conditions, under which AM proliferate long-term in liquid culture in the presence of granulocyte-macrophage colony-stimulating factor. This method can be used to obtain large numbers of AM for in vivo transplantation or for in vitro experiments with primary mouse macrophages.

Keywords: Alveolar; Bronchoalveolar lavage; Lungs; Macrophage; Primary cell culture; Self-renewal.

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Conflict of interest statement

Competing interests The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. Comparison of BAL conditions using pre-cooled or pre-warmed PBS with or without 2 mM EDTA.
Either 4 °C PBS without EDTA, 4 °C PBS with 2 mM EDTA and 0.5% FBS, or 37 °C PBS with 2 mM EDTA and 0.5% FBS was used. Numbers show the total amount of living cells (Trypan-Blue-negative) per BAL treatment per mouse. Each symbol denotes the mean cell count of 3 technical replicates of an individual mouse; horizontal lines indicate the mean, error bars show standard error of the mean (SEM); one-way ANOVA with Tukey’s multiple comparisons test; ns, non-significant.
Figure 2.
Figure 2.. FACS analysis of BAL AM.
A. Simple gating strategy for exclusion of doublets, dead cells. AM are SiglecF- and CD11c-positive. B-C. BAL cells harvested with pre-warmed BAL buffer containing EDTA are phenotypically not different from BAL cells harvested using pre-cooled PBS. Each symbol denotes 1 mouse. Typically, > 95% of BAL cells are AM. D. Viability analysis of BAL singlet cells assessed by staining with Zombie Violet Fixable Dye.
Figure 3.
Figure 3.. Representative images of AM culture within the first days after plating the cells.
A. AM culture with predominantly round-shaped cells that are partly floating and partly adherent on Day 1 after plating. B. Same culture as (A) on Day 2. C. Same culture as (A) on Day 4. D. Example for an AM culture with a large fraction of elongated, dark cells on Day 4 after plating. Arrowheads indicate dividing cells, 100× magnification.
See this image and copyright information in PMC

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