Immunology and Infection
Published: June 30th, 2016
A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.
A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.
Study of cultured macrophages is a useful model to understand the function of these cells in inflammation such as occurs in atherosclerotic plaques. When the research focus is on human diseases involving macrophages, it is useful to study primary human macrophages rather than non-human macrophages to avoid species differences. Also, the effects of cell transformation can be avoided by using primary macrophages rather than macrophage cell lines. For this purpose, macrophages differentiated from monocytes isolated from human blood serve as a means of obtaining primary human macrophages.
Tissue macrophages may be either resident within tissue....
Leukapheresis was carried out under a human subject's research protocol approved by a National Institutes of Health institutional review board.
1. Isolation and Cryopreservation of Monocytes
The viability of fresh or cryopreserved monocytes was greater than 95% as determined with Trypan Blue staining 9. Figure 1 and Figure 2 compare at lower and higher magnifications, respectively, the progress of fresh and cryopreserved (i.e., frozen) monocyte differentiation into macrophages. Note that the fresh compared with cryopreserved monocytes show a subpopulation of differentiating monocytes that do not spread but.......
Generating defined macrophage types can clarify some of the conflicting results obtained by investigators when studying macrophage biology. The use of various culture conditions and differentiation factors to generate primary human macrophages can lead to very different macrophage types, a fact that may not be appreciated by the researcher. For example, macrophages sometimes are generated from human monocytes using no serum, human serum alone, human serum supplemented with M-CSF, FBS alone, or FBS containing M-CSF or GM-.......
The Department of Transfusion Medicine, Clinical Center, National Institutes of Health, provided elutriated monocytes. This work was supported by the Intramural Research Program, National Heart, Lung, and Blood Institute, National Institutes of Health.....
|Cellbind 12-well culture plate
|CELLSTAR, T-75 flask, tissue culture treated
|Greiner Bio-One North America
|RPMI 1640 culture medium
|warmed to 37 °C
|Fetal bovine serum
|DPBS without Ca2+ and Mg2+
|50 ml polypropylene conical tube
|Neubauer-improved bright light hemocytometer
|Paul Marienfeld GmbH & Co. KG
|CoolCell LX cell freezing container
|other freezing containers also should be adequate for this step
|Liquid Nitrogen Storage System, CryoPlus 1
|any liquid nitrogen tank should be adequate
Copyright © 2024 MyJoVE Corporation. All rights reserved