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 density gradient medium (i.e., Percoll). Pre-chill on ice. 
	NOTE: IPS can be stored for up to 30 days at 4 &#176;C.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Anesthetize the mouse by using a mixture of ketamine/xylazine (90&#8722;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.</li>
	<li>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.</li>
	<li>Use forceps to gently grasp the heart by the right ventricle and lift it to the midline and slightly out of the chest.</li>
	<li>Insert a 23 G butterfly needle into the tip of the left ventricle, towards the aorta and hold firmly.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
</ol>
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.
<ol>
	<li>Use a pair of scissors to mince the tissues into 1&#8722;2 mm thick pieces.</li>
	<li>Cut the tip of a 1000 &#181;L 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.</li>
	<li>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.</li>
	<li>Centrifuge each sample for 5 min at 250 x g at 4 &#176;C.</li>
	<li>Prepare enzyme mix 1 of the neural tissue dissociation kit (NTDK; Table of Materials) by mixing 50 &#181;L of enzyme P with 1900 &#181;L of buffer X per sample. Warm enzyme mix 1 at 37 &#176;C in a water bath. Incubate enzyme mix 1 at 37 &#176;C for at least 10 min before use in order to allow for the full activation of the enzyme.</li>
	<li>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.</li>
	<li>Incubate the samples on a wheel or shaker for 15 min at 37 &#176;C.</li>
	<li>In the meanwhile, prepare enzyme mix 2 of the NTDK by mixing 10 &#181;L of enzyme A with 20 &#181;L of buffer Y per sample; pre-warm the solution at 37 &#176;C in a water bath.</li>
	<li>At the end of the incubation with enzyme mix 1, add 30 &#181;L of enzyme mix 2 to each sample.</li>
	<li>Gently mix the samples by pipetting up and down with a 1000 &#181;L pipette tip pre-rinsed with 1x HBSS.</li>
	<li>Incubate the sample on a wheel or shaker for 15 min at 37 &#176;C.</li>
	<li>After incubation, add 10 mL of ice-cold 1x HBSS to each tube to inactivate enzyme mix 1 and enzyme mix 2.</li>
	<li>Centrifuge each sample for 10 min at 320 x g at 4 &#176;C.</li>
	<li>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.</li>
	<li>Continue to section 5 for debris removal.</li>
</ol>
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.
<ol>
	<li>Pre-chill the glass mortar of the Dounce tissue grinder (Table of Materials) set on ice.</li>
	<li>Add 3 mL of pre-chilled 1x HBSS to the mortar.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>Continue to section 5 for debris removal.</li>
</ol>
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.
<ol>
	<li>Filter each sample through a 70 &#181;m 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Centrifuge samples for 15 min at 700 x g at 18 &#176;C making sure to set the acceleration of the centrifuge to 7 and the brake to 0. 
	NOTE: Centrifugation should take approximately 30 min.</li>
	<li>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.</li>
	<li>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 &#181;L of solution on top of the cell pellet to avoid the risk of inadvertently dislodging it.</li>
	<li>Add 1 mL of FACS BL, resuspend the pellet by pipetting up and down with a 1000 &#181;L pipette tip and transfer samples to 1.5 mL tubes.</li>
	<li>Centrifuge for 5 min at 450 x g at room temperature (RT).</li>
	<li>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).</li>
</ol>
6. Staining for flow cytometric evaluation of multiple cell types
<ol>
	<li>Resuspend the pellet obtained in step 5.9 with 350 &#181;L of FACS BL. Add Fc-block to each sample at a final concentration of 5 &#181;g/mL. 
	NOTE: At least 100 &#181;L of the sample should be used for one staining, to make sure to process enough cells to allow reliable analyses.</li>
	<li>Incubate the sample for 10 min at 4 &#176;C before proceeding with the staining.</li>
	<li>Prepare an antibody mix according to Table 1.</li>
	<li>Add antibody mix to each tube, vortex for 5 s and incubate the samples for 15 min at 4 &#176;C in the dark.</li>
	<li>Add 1 mL of PBS to each tube, vortex and centrifuge for 5 min at 450 x g at RT.</li>
	<li>Meanwhile prepare streptavidin mix according to Table 1.</li>
	<li>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.</li>
	<li>Vortex for 5 s and incubate the samples for 10 min at 4 &#176;C in the dark.</li>
	<li>Add 1 mL of PBS to each tube, vortex and centrifuge for 5 min at 450 x g at RT.</li>
	<li>Discard the supernatant and resuspend the pellet in FACS BL. Use 300 &#181;L of FACS BL for each 100 &#181;L of sample stained.</li>
	<li>Add 7-amino-actinomycin D (7-AAD) solution to each sample. Use 5 &#181;L of 7-AAD for each 300 &#181;L of sample prepared in step 6.10.</li>
	<li>Store samples at 4 &#176;C in the dark until cytofluorimetric analysis. Perform the analysis within 16 h from sample preparation, to guarantee &#62;60% cell viability.</li>
</ol>

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	<li>Deverman, B. E., Ravina, B. M., Bankiewicz, K. S., Paul, S. M., Sah, D. W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+neurological+disorders:+progress+and+prospects.">Gene therapy for neurological disorders: progress and prospects.</a> Nature Reviews Drug Discovery. 17 (9), 641-659 (2018).</li><li>Teleanu, D., Negut, I., Grumezescu, V., Grumezescu, A., Teleanu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomaterials+for+Drug+Delivery+to+the+Central+Nervous+System.">Nanomaterials for Drug Delivery to the Central Nervous System.</a> Nanomaterials. 9 (3), 371 (2019).</li><li>Chen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+Viral+Vectors+as+Neuroscience+Tools.">Recombinant Viral Vectors as Neuroscience Tools.</a> Current Protocols in Neuroscience. 87 (1), 67 (2019).</li><li>Alves, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultramicroscopy+as+a+novel+tool+to+unravel+the+tropism+of+AAV+gene+therapy+vectors+in+the+brain.">Ultramicroscopy as a novel tool to unravel the tropism of AAV gene therapy vectors in the brain.</a> Scientific Reports. 6 (1), 28272 (2016).</li><li>Peviani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+carrying+enhancer+elements+of+Hb9+promoter+drive+selective+transgene+expression+in+mouse+spinal+cord+motor+neurons.">Lentiviral vectors carrying enhancer elements of Hb9 promoter drive selective transgene expression in mouse spinal cord motor neurons.</a> Journal of Neuroscience Methods. 205 (1), 139-147 (2012).</li><li>Baumgarth, N., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+multicolor+flow+cytometry+for+immunophenotyping.">A practical approach to multicolor flow cytometry for immunophenotyping.</a> Journal of Immunological Methods. 243 (1-2), 77-97 (2000).</li><li>Sykora, M. 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The authors have nothing to disclose.

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 ...

simultaneous-flow-cytometric-characterization-multiple-cell-types

Research

JoVE Journal

Medicine

13.5K Views.  Dana-Farber/Boston Children's Cancer and Blood Disorders Center. De ontwikkeling van selectieve gen- en drugleveringsinstrumenten voor de behandeling van de neurodegeneratieve aandoeningen vereist de kwantificering en karakterisering van celtargeting, bij herbe toediening, of virale vectoren, of nanodeeltjes. Ze hebben doorvoer en multiplexing mogelijkheden van flow cytometrie, maakt de eenvoudige en gelijktijdige identificatie van meerdere celtypes uit de muis hersenen en het ruggenmerg. Het demonstreren van de procedure zal Francisco Javier Molina Esteve...

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

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

Immunology and Infection

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

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.

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 ...

Neuroscience

Assessing the Expression of Major Histocompatibility Complex Class I on Primary Murine Hippocampal Neurons by Flow Cytometry

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A well-studied function of major histocompatibility complex class I (MHCI) is the presentation of cell-derived peptides to the adaptive immune system, particularly in response to infection. More recently it has been shown that the expression of MHCI molecules on neurons can modulate activity-dependent changes in the synaptic connectivity during normal development and neurologic disorders. The importance of these functions to the brain health supports the need for a sensitive assay that readily detects MHCI expression on neurons. Here we describe a method for primary culture of murine hippocampal neurons and then assessment of MHCI expression by flow cytometric analysis. Murine hippocampus is microdissected from prenatal mouse pups at the embryonic day 18. Tissue is dissociated into a single cell suspension using enzymatic and mechanical techniques, then cultured in a serum-free media that limits growth of non-neuronal cells. After 7 days in vitro, MHCI expression is stimulated by treating cultured cells pharmacologically with beta interferon. MHCI molecules are labeled in situ with a fluorescently tagged antibody, then cells are non-enzymatically dissociated into a single cell suspension. To confirm the neuronal identity, cells are fixed with paraformaldehyde, permeabilized, and labeled with a fluorescently tagged antibody that recognizes neuronal nuclear antigen NeuN. MHCI expression is then quantified on neurons by flow cytometric analysis. Neuronal cultures can easily be manipulated by either genetic modifications or pharmacologic interventions to test specific hypotheses. With slight modifications, these methods can be used to culture other neuronal populations or to assess expression of other proteins of interest.

This protocol describes a method for culturing primary hippocampal neurons from embryonic mouse brain. The expression of major histocompatibility complex class I on the extracellular surface of cultured neurons is then assessed by flow cytometric analysis.

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (&gt;90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in ...

Mouse Brain Histone Modification Quantification Using Flow Cytometry

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Author Spotlight: Enhancements in Gene Expression Regulation Research

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

Obtaining Single-Cell Suspensions from Murine Brain and Skull Bone Marrow

Isolating Immune Cells from Mouse Brain and Skull

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

Author Spotlight: Investigating the Complex Immune Response Following Brain Ischemia

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only ...

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

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