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 ...

The development of selective gene and drug delivery tools to treat the neurodegenerative disorders, requires the quantification and characterization of cell targeting, upon re-administration, or viral vectors, or nanoparticles. They have throughput and multiplexing capabilities of flow cytometry, allows the straightforward and simultaneous identification of multiple cell types from the mouse brain and spinal cord. Demonstrating the procedure will be Francisco Javier Molina Estevez, a post-doc from Alexander's Laboratory.After harvesting the brain and spinal cord from an eight-week-old C57 Black 6 mouse, place the tissues into individual wells of a six-well plate, containing two milliliters of ice cold HPSS per well on ice. Divide each harvested tissue into two equal pieces. And use scissors to mince one half of each tissue sample into one to two millimeter thick pieces.When all of the tissues have been fragmented, pre-rinse a modified 1, 000 microliter pipette tip in HPSS, and use the pipette to transfer each tissue suspension into individual 15 milliliter conical tubes. Rinse the wells with an additional two milliliters of HPSS per well. And pull the washes in the corresponding 15 milliliter conical tubes.Sediment the samples by centrifugation. And mix 50 microliters of Enzyme P from a neural tissue dissociation kit with 1, 900 microliters of Buffer X from the kit per sample. Warm the enzyme mix at 37 degrees celsius for at least 10 minutes.And aspirate the supernatant from each tissue collection tube. When the enzyme mixture is ready, add 1.95 milliliters of the solution to each sample. And gently vortex to re-suspend the pellets.Next, incubate the samples on a wheel for 15 minutes at 37 degrees celsius, and mix 10 microliters of Enzyme A with 20 microliters of Buffer Y per sample. Prewarm the second enzyme solution at 37 degrees celsius, before adding 30 microliters of the solution to each tissue solution at the end of the shaking incubation. Use a 1, 000 microliter pipette tip, pre-rinsed with HPSS to gently mix each sample.And return the specimens to the wheel for 15 minutes at 37 degrees celsius. At the end of the incubation, arrest the enzymatic reactions with 10 milliliters of ice-cold HPSS. And sediment the samples by centrifugation.At the end of the centrifugation, re-suspend the pellets in seven milliliters of ice-cold HPSS per tube. And gently vortex each sample, before holding the tubes on ice. For tissue homogenization, add three milliliters of pre-chilled HPSS to the pre-chilled glass mortar of a dounce tissue grinder, and transfer the second half of one of the harvested brain or spinal cord tissue samples to the mortar.Gently macerate the tissue with 10 strokes of Pestle A, followed by 10 strokes of Pestle B.And transfer the homogenized mixture into a new 15 milliliter conical tube. Fill the tube to a final volume of 10 milliliters with pre-chilled HPSS for centrifugation. And re-suspend the pellet in seven milliliters of fresh HPSS with vortexing, before holding the sample on ice.For debris removal, add three milliliters of pre-chilled isotonic Percoll solution to each digested or homogenized sample. And gently vortex the samples to make sure they are homogeneously mixed. Next, centrifuge the samples, and carefully remove the whitish disc of debris and myelin floating at the surface of the solution.When the debris has been discarded, collect all but the last 100 microliters of the supernatant from each tube without disturbing the pellets. Re-suspend each pellet in one milliliter of FACS BL solution for transfer into individual 1.5 milliliter microcentrifuge tubes. After this integration with the Percoll solution, it's important to remove the debris disk and the supernatant very carefully without dislodging the cell pellet to avoid sample loss.After centrifugation, carefully aspirate the supernatant from each tube, and re-suspend the pellets in 350 microliters of fresh FACS BL per tube. For flow cytometric analysis of the isolated cell types, incubate the samples with five micrograms per milliliter of FC block per tube for 10 minutes at four degrees celsius before adding the appropriate antibody to each tube according to the standing protocol outlined in the table. Vortex each tube for five seconds to mix.And place the samples at four degrees celsius for 15 minutes protected from light. At the end of the incubation, wash the samples with one milliliter of PBS per tube, and centrifuge. Re-suspend the pellets in the appropriate volume of streptavidin per tube.After vortexing to mix, incubate the samples for 10 minutes at four degrees celsius protected from light. And wash the cells with one milliliter of PBS per tube as demonstrated. After discarding the supernatants, re-suspend the pellets in 300 microliters of fresh FACS BL per tube.And label each sample with five microliters of 7-AAD. Then store the samples at four degrees celsius protected from light until cytofluorometric analysis. The homogenization method produces a higher cell yield from both the brain and spinal cord overall, but the majority of the cells retrieved are typically dead, resulting in only and an approximately 14%healed of viable cells from the brain, and an approximately 10%healed from the spinal cord.In contrast, the papain digestion method results in an overall better preservation of cellular viability. Analysis of the brain and spinal cord cell suspensions with a nine color bi-flow cytometry, reveals the presence of CD45+CD11b+microglia, and macrophages. And CD45+CD11b-lymphocytes in both tissues.The CD45-cell populations can then be discriminated according to their positivity for astrocyte, or oligodendrocyte markers, or ferional and endothelial surface marker expression. Using the homogenization method, between 32 to 38%of the viable cells are of hematopoietic origin. While the enzymatic digestion method results in the acquisition of a very large fraction of a non-hematopoietic CD45-cells.Remarkably, CD45+CD11b+microglia and macrophages represent the most abundant viable cell fraction with the homogenization method. The digestion method, however, produces a more heterogeneous representation of cell types including astrocytes, oligodendrocytes, endothelial cells, and neurons. This protocol is versatile, and could be exploited for several downstream applications such as the quantification or isolation of specific cells subpopulations, primary culture, or biochemical, or RNA sequencing analysis.We have implemented this protocol to assess the gene expression and functional signatures of different CNS populations during this progression, or after treatment in a mouse model of endotrophic lateral sclerosis.

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