![]() ![]() For example, monocytes from heterozygous Cx3cr1 GFP/+ mice display an altered phenotype in at least some situations ( Combadière et al., 2003). Although this is generally true, there can be subtle gene dosage effects on cell function that may not be detected unless specifically scrutinized. This assumes bi-allelic expression (both alleles are expressed in each cell) and haplosufficiency (half the amount of mRNA for the endogenous gene is sufficient for function). Knock-in mice made using this method are typically used as heterozygous mice, where one allele encodes the fluorescent protein and the other allele the endogenous protein. Traditionally, knock-in mice are made by inserting the cDNA for a fluorescent protein into the natural gene locus, using gene targeting and homologous recombination to replace all or part of the endogenous allele with the cDNA for a fluorescent protein. To tag a specific subset of cells, it is important to choose both the right gene on which to report, as well as the right type of construct. Multiple reporters of different fluorescent proteins can be combined, as long as the colors can be spectrally separated ( Feng et al., 2000). There are a wide range of reporter mice available that are suitable for intravital imaging of myeloid cells, and many have been tested in atherosclerosis ( Table 1). An important advantage of genetically labeled cells is that they usually continue to express the fluorescent proteins after long periods of cell culture, or after being adoptively transferred to another mouse. Also, the commonly used C57BL/6 recipient mice can reject cells labeled with dsRed protein and some of its derivatives ( Davey et al., 2013). However, there can be immunological barriers to bone marrow transplantation. In some cases, bone marrow transplantation ( Stark et al., 2013) or adoptive transfer ( Shaked et al., 2015) can be used to label myeloid cells without crossing mice. However, once a line is created, no additional work is needed to label every mouse. ( Abe and Fujimori, 2013) Engineering reporter mice can be expensive, and it is time-consuming to cross them into other mouse strains. GFP and YFP are the most commonly used labels, though mice with cyan fluorescent protein (CFP), or various red fluorescent proteins (RFPs), are available. Genetic labeling techniques rely on constructs that report the expression of a gene via a fluorescent protein (FP). This review discusses the common methods for each of these steps for imaging myeloid cells, as well as the necessity of incorporating other techniques towards the best interpretation of the data. Each of these applications share some common traits: the cells of interest must be labeled, the tissue must be stabilized, and the data must be quantitatively analyzed. Myeloid cells have been imaged in a variety of tissues, including the spinal cord and brain ( Kim et al., 2009), liver ( Geissmann et al., 2005 Egen et al., 2011), kidney ( Soos et al., 2006), spleen ( Swirski et al., 2009), ear ( Auffray et al., 2007), intestine ( Chieppa et al., 2006), and recently atherosclerotic arteries ( Drechsler et al., 2010 Chèvre et al., 2014 McArdle et al., 2015). Intravital imaging of dendritic cells was first reported in lymph nodes ( Mempel et al., 2004 Germain et al., 2006). ![]() Cell motion ( Germain et al., 2012), cell proliferation ( Stoll et al., 2002), cell death ( Mempel et al., 2006), and cell–cell interactions ( Cahalan and Parker, 2008) have been previously observed. Intravital imaging has been used to visualize diverse leukocyte behaviors in a variety of contexts. ![]()
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