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

Summary

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.

Abstract

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.

Introduction

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 approaches^1,2,3. 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 markers^4,5. 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 cells^9,10,11; 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 °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 selection^9,12, 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).

Protocol

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

1. Prepare 1x Hank’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.
2. Prepare isotonic Percoll solution (IPS) by mixing 10x sterile HBSS 1:10 with density gradient medium (i.e., Percoll). Pre-chill on ice.
   NOTE: IPS can be stored for up to 30 days at 4 °C.
3. Prepare flow cytometry (FACS) blocking (BL) solution (1% bovine serum albumin [BSA], 5% fetal bovine serum [FBS] in phosphate-buffered saline [PBS]). Pre-chill on ice.

2. Animal euthanasia by intracardiac perfusion and tissue dissection

NOTE: Eight-week-old C57BL/6J mice, either sex, were used in the experiments. Perfusion with PBS solution is performed to eliminate blood contamination from organs, before proceeding with tissue digestion.

1. Anesthetize the mouse by using a mixture of ketamine/xylazine (90−200 mg/kg ketamine, 10 mg/kg xylazine). Place the mouse on its back and tape each limb down to the support. Verify adequate depth of anesthesia by checking the withdrawal reflex.
2. Make a midline skin incision at the level of the thoracic inlet to expose the sternum. Use forceps to grasp the tip of the sternum, then make one 1 cm incision on each side of the rib cage. Finally cut through the diaphragm and open the sternum widely enough to visualize the heart.
3. Use forceps to gently grasp the heart by the right ventricle and lift it to the midline and slightly out of the chest.
4. Insert a 23 G butterfly needle into the tip of the left ventricle, towards the aorta and hold firmly.
5. Start the perfusion with 1x PBS. Pierce through the right auricle using scissors to allow the perfusate to exit the circulation. Set the flow rate of PBS at 3 mL/min. Perfuse with at least 15 mL of 1x PBS to ensure tissues are clear.
   NOTE: Blanching of the liver and mesenteric blood vessels are signs of good perfusion. If necessary, the volume of prefusion can be increased up until the fluid exiting the heart is clear of blood, at which point the flush line can be stopped.
6. After perfusion, sever the brain from the spinal cord and remove the brain from the skull with scissors and forceps. Remove the fur to increase visibility and control during the dissection and to avoid carrying over hair contaminants. Flush the spinal cord out of its column by using a 3 mL syringe filled with PBS.
7. Transfer each tissue in a well of a 6-well multi-well plate prefilled with 2 mL of ice-cold 1x HBSS and keep on ice until digestion.
8. Divide the brain and the spinal cord into two halves, along the longitudinal line.
   NOTE: One half of each tissue is homogenized (see sections below) to allow flow cytometric analyses; the other half can be assigned to different processing for alternative analyses (e.g., dipped in paraformaldehyde fixative solution for histology).

3. Enzymatic digestion of brain and spinal cord

NOTE: Volumes described in this section are enough for digestion of one-half brain or spinal cord.

1. Use a pair of scissors to mince the tissues into 1−2 mm thick pieces.
2. Cut the tip of a 1000 µL pipette with a pair of scissors to make it sufficiently large to allow the collection of the tissue pieces. Pre-rinse the pipette tip with 1x HBSS. Then use the pipette to transfer the 2 mL of HBSS solution containing the minced tissue to a 15 mL conical tube.
   NOTE: Pre-rinsing of the pipette tip is important to prevent stickiness of the tissue pieces inside the tip.
3. Wash the well with additional 2 mL of ice-cold 1x HBSS and transfer the solution to the corresponding 15 mL conical tube containing the tissue pieces.
4. Centrifuge each sample for 5 min at 250 x g at 4 °C.
5. Prepare enzyme mix 1 of the neural tissue dissociation kit (NTDK; Table of Materials) by mixing 50 µL of enzyme P with 1900 µL of buffer X per sample. Warm enzyme mix 1 at 37 °C in a water bath. Incubate enzyme mix 1 at 37 °C for at least 10 min before use in order to allow for the full activation of the enzyme.
6. Aspirate the supernatant from the 15 mL conical tube and add 1.95 mL of enzyme mix 1 to each sample. Gently vortex to make sure the pellet is resuspended.
7. Incubate the samples on a wheel or shaker for 15 min at 37 °C.
8. In the meanwhile, prepare enzyme mix 2 of the NTDK by mixing 10 µL of enzyme A with 20 µL of buffer Y per sample; pre-warm the solution at 37 °C in a water bath.
9. At the end of the incubation with enzyme mix 1, add 30 µL of enzyme mix 2 to each sample.
10. Gently mix the samples by pipetting up and down with a 1000 µL pipette tip pre-rinsed with 1x HBSS.
11. Incubate the sample on a wheel or shaker for 15 min at 37 °C.
12. After incubation, add 10 mL of ice-cold 1x HBSS to each tube to inactivate enzyme mix 1 and enzyme mix 2.
13. Centrifuge each sample for 10 min at 320 x g at 4 °C.
14. Discard the supernatant; add ice-cold 1x HBSS to each tube up to a final volume of 7 mL and gently resuspend the pellet by vortexing.
15. Continue to section 5 for debris removal.

4. Mechanical homogenization of brain and spinal cord

NOTE: Volumes described in this section are enough for homogenization of one-half brain or spinal cord. The protocol described in this section can be used as a method alternative to the one described in section 3, depending on user need as discussed below.

1. Pre-chill the glass mortar of the Dounce tissue grinder (Table of Materials) set on ice.
2. Add 3 mL of pre-chilled 1x HBSS to the mortar.
3. Transfer the tissue (brain or spinal cord) from the well of the 6-well plate into the glass mortar making sure it is dipped in 1x HBSS and sits at the bottom of the mortar.
4. Gently smash the tissue with 10 strokes of pestle A followed by 10 strokes of pestle B. Transfer the homogenized mix into a new 15 mL conical tube.
5. Fill the tube to a final volume of 10 mL by using pre-chilled 1x HBSS and centrifuge for 10 min at 320 x g at 4 °C.
6. Aspirate the supernatant and add ice-cold 1x HBSS to each tube up to a final volume of 7 mL and gently resuspend the pellet by vortexing.
7. Continue to section 5 for debris removal.

5. Debris removal

NOTE: Removal of debris, composed mainly of undigested tissue and myelin sheaths, is a critical step to allow efficient staining of the tissue homogenate for subsequent flow cytometric analyses.

1. Filter each sample through a 70 µm cell strainer to remove any undigested tissue chunk. This step is particularly important especially when working with spinal cord tissues since these samples are more likely to contain undigested nerve fragments or meninges that could affect the subsequent steps.
2. Make sure that the final volume is 7 mL in each sample tube. If not, fill with ice-cold 1x HBSS up to 7 mL.
3. Add 3 mL of pre-chilled IPS to each sample to make a final volume of 10 mL of a solution containing density gradient medium at 30% final concentration. Gently vortex the samples to make sure they are homogenously mixed.
4. Centrifuge samples for 15 min at 700 x g at 18 °C making sure to set the acceleration of the centrifuge to 7 and the brake to 0.
   NOTE: Centrifugation should take approximately 30 min.
5. Delicately remove the samples from the centrifuge.
   NOTE: A whitish disk composed of debris and myelin should be visible floating on the surface of the solution. A pellet (containing the cells of interest) should be visible at the bottom of the tube.
6. Carefully aspirate all the whitish disk of debris and then the rest of the supernatant making sure not to dislodge the pellet. Leave about 100 µL of solution on top of the cell pellet to avoid the risk of inadvertently dislodging it.
7. Add 1 mL of FACS BL, resuspend the pellet by pipetting up and down with a 1000 µL pipette tip and transfer samples to 1.5 mL tubes.
8. Centrifuge for 5 min at 450 x g at room temperature (RT).
9. Gently aspirate the supernatant and resuspend the pellet in appropriate buffer compatible with downstream analyses (see section 6 for the protocol used for flow cytometric evaluation of multiple cell types).

6. Staining for flow cytometric evaluation of multiple cell types

1. Resuspend the pellet obtained in step 5.9 with 350 µL of FACS BL. Add Fc-block to each sample at a final concentration of 5 µg/mL.
   NOTE: At least 100 µL of the sample should be used for one staining, to make sure to process enough cells to allow reliable analyses.
2. Incubate the sample for 10 min at 4 °C before proceeding with the staining.
3. Prepare an antibody mix according to Table 1.
4. Add antibody mix to each tube, vortex for 5 s and incubate the samples for 15 min at 4 °C in the dark.
5. Add 1 mL of PBS to each tube, vortex and centrifuge for 5 min at 450 x g at RT.
6. Meanwhile prepare streptavidin mix according to Table 1.
7. Discard the supernatant and resuspend the pellet in the streptavidin mix prepared in step 6.6. For each sample, use the same volume as the one used for the staining in step 6.4.
8. Vortex for 5 s and incubate the samples for 10 min at 4 °C in the dark.
9. Add 1 mL of PBS to each tube, vortex and centrifuge for 5 min at 450 x g at RT.
10. Discard the supernatant and resuspend the pellet in FACS BL. Use 300 µL of FACS BL for each 100 µL of sample stained.
11. Add 7-amino-actinomycin D (7-AAD) solution to each sample. Use 5 µL of 7-AAD for each 300 µL of sample prepared in step 6.10.
12. Store samples at 4 °C in the dark until cytofluorimetric analysis. Perform the analysis within 16 h from sample preparation, to guarantee >60% cell viability.

Results

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 ≥ 6), spl...

Discussion

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 CNS^5,13, or to provide insights on morphological and molecular changes occurring in specific cell types during a pathology or upon pharmacologic...

Materials

List of materials used in this article

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