Name: analysis of immune cells in single sciatic nerves and dorsal root ganglion from a single mouse using flow cytometry
Uploaded: 2017-12-06T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry at JoVE.com

This study was supported by the Deutsche Forschungsgemeinschaft (DFG; SFB1118). The authors would like to thank Axel Erhardt for performing most of the dissection and helpfully transmitting this knowledge, and Dr. Volker Eckstein for assisting in the technical aspect of the flow cytometer.

Wild-type C57BL/6 mice (Males; 10-12 weeks) were kept in standard 12 h light/dark cycle and were provided free access to standard chow diet and water. All animal experiments were conducted in accordance to the relevant guidelines by the local Animal Care and Use Committee and approved by the local Animal Care and Use Committee at the regional authority in Karlsruhe, Germany (G216/10).
1. Perfusion of the Mouse
<ol>
	<li>Sacrifice the mouse (C57BL/6; Males; 10-12weeks) using CO2 or...

<ol>
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Sciatic nerves contain a large proportion of lipids, such as cholesterol, due to the content of myelin around the axons. Since the properties of lipids change with temperature, different results may be obtained at different temperatures. To ensure cell preservation, all steps in this protocol after the digestion were performed on ice. Whilst consistency is recommended for the sake of reproducibility of the results, it may be possible to increase yield by performing the steps 3.6 and 3.7 at room temperature. If the nerves...

Immune cells which enter the PNS from the circulation, as defined by the expression of CD45 and CD11b, help to maintain the integrity of the nerve and play a role both in regeneration and degeneration1. Macrophages (defined by their expression of CD68 in mouse) can be skewed towards an inflammatory phenotype, expressing more MHC class II and CD86 on their surface (M1), or towards an anti-inflammatory phenotype, expressing more intracellular CD206 (M2)2. Skewing of the macrophage phenotype is a dynamic process regulated through Akt signaling3, reflecting the different tasks of macrophages in the defense against pathogens (M1) and the role in tissue regeneration (M2). Regeneration of an injured nerve first requires phagocytosis of myelin debris by macrophages in the nerve4,5, and anti-inflammatory (CD68+CD206+) M2 macrophages have been shown to promote axon outgrowth in the PNS6. Reduced recruitment or macrophages to the PNS, or impaired capacity for phagocytosis may result in impaired regeneration and maintenance of nerve integrity. Inflammatory M1 macrophages, expressing MHC class II, are less capable of phagocytosis than M2 macrophages, and neuronal inflammation is implicated in the pathogenesis of several neurodegenerative diseases7.
The changes that occur to the nerve resident immune system as a consequence of damage may be quantitative, manifesting in loss of CD45+ leukocytes (or increased infiltration of CD45+ leukocytes in the case inflammation), or qualitative, such as change of macrophage phenotype from M2 to M1 phenotype. The immune cells of the PNS have traditionally been analyzed by means of IF, using frozen sections or paraffin-embedded material8. IF is required for determining localization of the cells of interest. However, quantification using IF often relies on counting a relatively small number of cells in a narrow section of the tissue, making quantification unreliable and vulnerable to selection bias. For identification of specific subsets of immune cells, the simultaneous detection of extracellular and/or intracellular markers is required, whilst determination of the macrophage phenotype requires at least at least two markers, specifically CD206 and MHC class II. As most commonly available microscopes are limited to at least two-color channels, such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE), the characterization of the specific subsets of immune cells by IF can be restrictive and incomplete, requiring the need to have multiple slides, derived from the same area of interest, which are stained and analyzed in parallel. This time-consuming aspect therefore does not necessary lend itself to the analysis of large sample sets. Furthermore, as most of the markers of interest are extracellular, the detection in tissue, which has either been embedded in paraffin or cryoconserved, can be problematic due to the disruption of membrane integrity and the masking of epitopes, as well as the loss of the antigens of interest from the use of solvents, such as acetone and methanol9.
In contrast, flow cytometry, which measures optical and fluorescence characteristics of single cells in suspension as they pass through a beam of light, provides a more practical and comprehensive means for analysis of the cell populations. Flow cytometry, rather than producing a digital image of the tissue, provides an automated quantification of set parameters, which include a cell&#39;s relative size and reflective index, referred to as the forward scatter (FSC), granularity/internal complexity or side-scatter (SSC), and relative fluorescence intensity, providing that the cell has been labeled with an appropriate fluorophore, such as a conjugated antibody. A typical flow cytometer consists of two, air-cooled lasers; an argon laser produces blue light at 488 nm and a helium-neon laser produces light at 633 nm. This combination allows for the detection and measurement, simultaneously, of at least five targets either on the surface or within the intracellular compartment. More advanced flow cytometers can consist of multiple lasers, which allow for the detection of up to eight different fluorochromes at once, providing that the peak emission wavelengths of the selected fluorochromes do not overlap significantly.
For analyzing by flow cytometry, the tissue of interest must first be enzymatically digested, generally with collagenase, to generate a single-cell suspension. The analysis of murine sciatic nerves has previously been difficult due to the small amount of tissue obtained from each mouse. In addition, the high fat content of the myelin around the axons hampers cell recovery and produces large amounts of debris. The method described herein for sciatic nerve preparation and digestion was adapted from the Schwann cell isolation protocol of Barrette et al.7, and aims to isolate enough cells from nerves of individual mice for flow cytometry analysis, in order to reduce variation between mice. Using DAPI for the identification of single cells in the raw tissue digest circumvents the need to remove axon debris, which commonly leads to cell loss. Washing several times with a detergent-rich buffer aids in the release of cells trapped within the fatty debris, thereby increasing the yield. Digestion of both full-length sciatic nerves from a single mouse according to this protocol generates &#8805;30,000 single nucleated events and at least 3 times that number was retrieved from the DRG. The proportion of CD45+ leukocytes was approximately 5% of total cell content in the sciatic nerve digest and approximately 5-10% in the DRG digest. The majority of the CD45+ cells in the sciatic nerve expressed the macrophage markers, CD68 and CD206.

<table>
	<tbody>
		<tr>
			<td>C57BL/6 Mouse</td>
			<td>Charles River</td>
			<td>C57BL/6NCrl</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (+1g/L glucose, Glutamine, Pyruvate)</td>
			<td>Thermo</td>
			<td>31885023</td>
			<td>The source of this material is not important</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type 4</td>
			<td>Worthington Biochemical Corp., US</td>
			<td>LS004188</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease (DNase) I from bovine pancreas I</td>
			<td>Sigma-Aldrich</td>
			<td>DN25-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Calf Serum (FCS), heat inactivated</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody against CD11b-PerCP/Cy5.5</td>
			<td>Biolegend</td>
			<td>101228</td>
			<td>Clone M1/70</td>
		</tr>
		<tr>
			<td>Antibody against MHC class II, biotinylated</td>
			<td>Biolegend</td>
			<td>107603</td>
			<td>Clone M5/114.15.2</td>
		</tr>
		<tr>
			<td>Antibody against CD45-A647</td>
			<td>Biolegend</td>
			<td>107603</td>
			<td>Clone 30-F11</td>
		</tr>
		<tr>
			<td>Antibody against CD68-APC</td>
			<td>Biolegend</td>
			<td>137007</td>
			<td>Clone FA/11</td>
		</tr>
		<tr>
			<td>Antibody against CD206-PE</td>
			<td>Biolegend</td>
			<td>141706</td>
			<td>Clone C068C2</td>
		</tr>
		<tr>
			<td>Antibody against F4/80-PE/Cy7</td>
			<td>Biolegend</td>
			<td>123113</td>
			<td>Clone BM8</td>
		</tr>
		<tr>
			<td>Streptavidin-PE/Cy7</td>
			<td>Biolegend</td>
			<td>405206</td>
			<td>Used to detect MHCII-biotin with CD45-A647 and CDllb-PerCP/Cy5.5</td>
		</tr>
		<tr>
			<td>Streptavidin-PerCP</td>
			<td>Biolegend</td>
			<td>405213</td>
			<td>Used to detect MHCII-biotin with CD68-APC and F4/80-PE/Cy7</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-1MG</td>
			<td>Dilute in PBS to a 50x and store at 4 &#176;C in the dark</td>
		</tr>
		<tr>
			<td>Cell dissociation sieve - tissue grinder kit</td>
			<td>Sigma-Aldrich</td>
			<td>CD1</td>
			<td></td>
		</tr>
		<tr>
			<td>V-Shaped, 96-well plates</td>
			<td>Greiner/Sigma</td>
			<td>M8185</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml polystryene round-bottom tube, 12x75mm</td>
			<td>BD Biosciences</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>60x15mm petri dish</td>
			<td>Greiner/Sigma</td>
			<td>Z643084</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSR II Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of immune cells in single sciatic nerves and dorsal root ganglion from a single mouse using flow cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Cell suspensions from both full-length sciatic nerves and all major DRG were prepared, according to the protocol, from six healthy C57BL/6 mice and divided into three equal aliquots for staining. Counter staining with DAPI, which stains DNA, allows for the detection of a single cell population (Figure 1A-B, upper panel). Washing out the DAPI, prior to analysis by flow cytometry, decreased to an extent the fluorescent intensit...

Watch this Scientific Journal Video about Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry at JoVE.com

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

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