Name: simultaneous flow cytometric characterization of multiple cell types retrieved from mouse brain/spinal cord through different homogenization methods
Uploaded: 2019-11-19T01:00:00
Description: Watch this Scientific Journal Video about Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods at JoVE.c

This study was funded by Boston Children&#8217;s Hospital start-up funds to A.B., ALSA grant nr. 17-IIP-343 to M.P., and the Office of the Assistant Secretary of Defense for Health Affairs through the Amyotrophic Lateral Sclerosis Research Program under Award No. W81XWH-17-1-0036 to M.P. We acknowledge DFCI Flow Cytometry Core for technical support.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Dana Farber Cancer Institute (protocol number 16-024).
1. Preparation of solutions needed for the experiment
<ol>
	<li>Prepare 1x Hank&#8217;s balanced salt solution (HBSS) by diluting 10x HBSS with sterile water. Pre-chill the solution on ice. At least 25 mL of solution are needed for each sample.</li>
	<li>Prepare isotonic Percoll solution (IPS) by mixing 10x sterile HBSS 1:10 with d...

<ol>
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Herein we describe a protocol for the co-purification and concurrent flow cytometric analysis of some of the most relevant CNS cells from mouse brain and spinal cord. Traditionally, histological analyses have been applied to describe the distribution of nanoparticles or the transduction efficiency of viral vectors in the CNS5,13, or to provide insights on morphological and molecular changes occurring in specific cell types during a pathology or upon pharmacologic...

Gene and drug delivery technologies (such as viral vectors and nanoparticles) have become a powerful tool that can be applied to gain better insights on specific molecular pathways altered in neurodegenerative diseases and for development of innovative therapeutic approaches1,2,3. Optimization of these tools relies on quantification of: (1) the extent of penetration in the CNS upon different routes of administration and (2) targeting of specific cell populations. Histological analyses are usually applied to visualize fluorescent reporter genes or fluorescently-tagged nanoparticles in different CNS areas and across different cell types, identified by immunostaining for specific cell markers4,5. Even though this approach provides valuable information on the biodistribution of the administered gene or drug-delivery tools, the technique can be time-consuming and labor-intense since it requires: (1) tissue fixation, cryopreservation or paraffin-embedding and slicing; (2) staining for specific cellular markers sometimes requiring antigen retrieval; (3) acquisition by fluorescence microscopy, which usually allows the analysis of a limited number of different markers within the same experiment; (4) image processing to allow proper quantification of the signal of interest.
Flow cytometry has become a widely used technique which takes advantage of very specific fluorescent markers to allow not only a rapid quantitative evaluation of different cell phenotypes in cell suspensions, based on expression of surface or intracellular antigens, but also functional measurements (e.g., rate of apoptosis, proliferation, cell cycle analysis, etc.). Physical isolation of cells through fluorescent activated cell sorting is also possible, allowing further downstream applications (e.g., cell culture, RNAseq, biochemical analyses etc.)6,7,8.
Tissue homogenization is a critical step necessary to obtain a single cell suspension to allow reliable and reproducible downstream flow cytometric evaluations. Different methods have been described for adult brain-tissue homogenization, mainly with the aim to isolate microglia cells9,10,11; they can be overall classified in two main categories: (1) mechanical dissociation, which uses grinding or shearing force through a Dounce homogenizer (DH) to rip apart cells from their niches and form a relatively homogenized single cell suspension, and (2) enzymatic digestion, which relies on incubation of minced tissue chunks at 37 &#176;C in the presence of proteolytic enzymes, such as trypsin or papain, favoring the degradation of the extracellular matrix to create a fairly homogenized cell suspension12.
Regardless of which method is utilized, a purification step is recommended after tissue homogenization to remove myelin through centrifugation on a density gradient or by magnetic selection9,12, before moving to the downstream applications.
Here, we describe a tissue processing method based on papain digestion (PD) followed by purification on a density gradient, optimized to obtain viable heterogeneous cell suspensions from mouse brain or spinal cord in a time-sensitive manner and suitable for flow cytometry. Moreover, we describe a 9-color flow cytometry panel and the gating strategy we adopted in the laboratory to allow the simultaneous discrimination of different CNS populations, live/dead cells or positivity for fluorescent reporters such as green fluorescent protein or rhodamine dye. By applying this flow cytometric analysis, we can compare different methods of tissue processing, i.e., PD versus DH, in terms of preservation of cellular viability and yields of different cell types.
The details we provide herein can instruct decision on the homogenization protocol and the antibody combination to use in the flow cytometry panel, based on the specific cell type(s) of interest and the downstream analyses (e.g., temperature-sensitive applications, tracking of specific fluorescent markers, in vitro culture, functional analyses).

<table>
	<tbody>
		<tr>
			<td>10X HBSS (Calcium, Magnesium chloride, and Magnesium Sulfate-free)</td>
			<td>Gibco</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>70 mm Cell Strainer</td>
			<td>Corning</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>ACSA/ACSA2 anti-mouse antibody</td>
			<td>Miltenyi Biotec</td>
			<td>130-117-535</td>
			<td>APC conjugated</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A9647-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b rat anti-mouse antibody</td>
			<td>Invitrogen</td>
			<td>47-0112-82</td>
			<td>APC-eFluor 780 conjugated</td>
		</tr>
		<tr>
			<td>CD31 rat anti-mouse antibody</td>
			<td>BD Bioscience</td>
			<td>562939</td>
			<td>BV421 conjugated</td>
		</tr>
		<tr>
			<td>CD45 rat anti-mouse antibody</td>
			<td>Biolegend</td>
			<td>103138</td>
			<td>Brilliant Violet 510 conjugated</td>
		</tr>
		<tr>
			<td>CD90.1/Thy1.1 rat anti-mouse antibody</td>
			<td>Biolegend</td>
			<td>202518</td>
			<td>PE/Cy7 conjugated</td>
		</tr>
		<tr>
			<td>CD90.2/Thy1.2 rat anti-mouse antibody</td>
			<td>Biolegend</td>
			<td>1005325</td>
			<td>PE/Cy7 conjugated</td>
		</tr>
		<tr>
			<td>Conical Tubes (15 mL)</td>
			<td>CellTreat</td>
			<td>229411</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Tubes (50 mL)</td>
			<td>CellTreat</td>
			<td>229422</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Tissue Grinder set (Includes Mortar as well as Pestles A and B)</td>
			<td>Sigma-Aldrich</td>
			<td>D9063-1SET</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc (CD16/CD32) Block rat anti-mouse antibody</td>
			<td>BD Pharmingen</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Benchmark</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Tissue Dissociation Kit (P)</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-628</td>
			<td></td>
		</tr>
		<tr>
			<td>O4 anti mouse/rat/human antibody</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-895</td>
			<td>Biotin conjugated</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>10266569</td>
			<td>sold as not sterile reagent</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Sigma</td>
			<td>65455529</td>
			<td>sterile reagent (to be used for applications requiring sterility)</td>
		</tr>
		<tr>
			<td>Percoll PLUS</td>
			<td>Sigma</td>
			<td>GE17-5445-01</td>
			<td>reagent containing very low traces of endotoxin</td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Invitrogen</td>
			<td>S3258</td>
			<td>Alexa Fluor 680 conjugated</td>
		</tr>
	</tbody>
</table>

simultaneous flow cytometric characterization of multiple cell types retrieved from mouse brain/spinal cord through different homogenization methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We compared two different homogenization methods (DH versus PD) applied to mouse brain and spinal cord, to test the efficiency in retrieving different viable cell types suitable for downstream applications. To do so, we exploited a 9-color flow cytometry panel designed to characterize, in the same sample, different CNS cell types including microglia, lymphocytes, neurons, astrocytes, oligodendrocytes and endothelium.
Brain and spinal cord tissues were retrieved from different mice (n &#8805; 6...

Watch this Scientific Journal Video about Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods at JoVE.c

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

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