Abstract
The generation of mast cells for in vitro studies comes from a variety of sources including mast cell lines (MC/9) (McCurdy et al., 2001), bone marrow-derived mast cells (BMMCs) (Supajatura et al., 2001), skin-derived mast cells (FSMCs) (Matsushima et al., 2004), peritoneal-derived mast cells (PMCs) (Hochdorfer et al., 2011) and peritoneal cell-derived cultured mast cells (PCMCs) (Vukman et al., 2012). BMMCs are generally used for in vitro studies because of the high yield of mast cells generated and also because they can be generated from knockout and transgenic mice making this a good source to examine specific factors important for mast cell function. Due to the large yield of cells generated they are the cells of choice for reconstitution studies in mast cell knockout mice (Sur et al., 2007). Furthermore, they are more responsive to both allergic and non-allergic stimuli when compared to mast cell lines. The major disadvantage of BMMCs is that they are not fully matured when compared to mast cells generated or obtained from other sources. For example, compared to PCMCs [see the protocol “Isolation and Culture of Peritoneal Cell-derived Mast Cells” (Vukman et al., 2014)], BMMCs express a restricted range of TLRs and cytokines when stimulated with TLR ligands (Mrabet-Dahbi et al., 2009). The different sources of mast cells can display phenotypical and functional differences and therefore it is important that when designing an experiment the correct cellular source is obtained. Here, we describe a protocol for the isolation and culture of murine mast cells from mouse bone marrow.
Keywords: Mast cells, Cell culture, Primary cells, Bone marrow, WEHI-3
Materials and Reagents
Equipment
Procedure
Note: All procedures are performed in a sterile environment in a class II safety cabinet.
Recipes
Acknowledgments
The protocol described here was adapted from Sur et al. (2007) and Vukman et al. (2012).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Hi, After the peritoneal lavage if you leave peritoneal cells in medium (withouth WEHI-3 condotioned medium) for few hours, macrophage will adhere and you can use them. I think it should work from the mast cell culture as well.
You can do a western, but I think with that method you can only prove that you have mast cells in your cell culture, but are not able to check purity. I think Kimura stain is the easiest way to check purity. I ususally use facs, because it is very specific and reliable.
Kimura stain contains toluidine blue and it is often used to identify mast cells, because they stain heparin in their cytoplasmic granules. I tried it with some other bone marrow-derived cells and it was specific for mast cells. (Krisztina)
When feeding the cells it is important to take only the non-adherent cells. The cells adhered to the flask are not mast cells, so try not to get them in suspension. The mast cells yield in the begining is very low, but it will be 30-40 million by passage 9, and there will be less and less adherent cells. We also use flasks for non-adherent cells (cat.no. in the protocol).
We did not see any difference within BMMCs from old and young mice. In other labs they prefer to use old (few month old) mice.