Name: Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients (Video) | JoVE Journal
Uploaded: 2011-02-02T17:00:00.000+00:00
Duration: 9 min 58 s
Description: Learn to isolate mononuclear cells from mouse brain and spinal cord using discontinuous Percoll gradients. This rapid protocol enables flow cytometric phenotyping of CNS-infiltrating immune populations.

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5 minutes

This work was supported by the National Multiple Sclerosis Society (TA 3021-A1/T to AEC) and the University of Texas at San Antonio.

Preparation of reagents
Prepare stock isotonic percoll (SIP), 4 ml per brain, by mixing 9 parts of percoll with one part of 10X HBSS without Ca++ and Mg++.
Prepare SIP at 70% in 1X HBSS without Ca++ and Mg++, 2 ml per brain dispensed into a 15 ml polypropylene conical tube.
Note: It is recommended that percoll should be used at room temperature, if used cold, cells tend to clump and cell separation is less efficient.
Tissue collection and homogenization
Anesthetize mice and perfuse through the left ventricle with ice-cold 1X HBSS. Dissect brain and spinal cord and maintain in RPMI without phenol red until all mice are sacrificed.
Place tissues in 7 or 15 ml dounce homogenizer containing 3 ml of RPMI, gently make a cell suspension with a loose-fitting pestle (A size) followed by a tigh-fitting pestle (B size) to further dissociate the tissues. Complete the volume of the cell suspensions to 7 ml with RPMI.
Gradient set up
Add 3 ml of SIP to the cell suspension to make a final 30% SIP
Slowly layer the 10 mL cell suspension on top of the 70% SIP. This is the most critical step of the procedure, use a pipette-aid set in the gravity mode avoiding mixing of the 70% and 30% solutions. A very clear flat line should be visible at the 70%-30% junction.
Centrifuge 30 min at 500G 18°C. Make sure centrifuge will stop with minimal or no brake so that the interphase is not disturbed.
Using a transfer pipet, gently remove the layer of debris from the top of the tube and collect 2.0-3.0 ml of the 70%-30% interphase into a clean conical tube containing 8 ml of 1X HBSS. Ensure that the percoll containing the interphase is diluted about three fold, mix a few times by inversion and centrifugre 7 min at 500G at 18°C.
Resuspend pellet in 1 ml of cell staining buffer and transfer it to a 1.5 ml tube and wash one more time using a micro-centrifuge at 10,000G 1 min at 4°C.
Antibody staining
Resuspend pellets in 50 μl of purified antimouse CD16/CD32 diluted 1:200 in cell staining buffer to block the Fc binding sites. Incubate over ice for 10 min. Count cells using a hemocytometer.
Add 50 μl of antibody cocktail mix and incubate on ice for 30 min. Each antibody must be first titrated to assess optimal dilution. Use appropriate fluorochrome combinations chosen according the capabilities of laser and filter settings of your particular flow cytometer.
Wash cells in cell staining buffer, resuspend pellets in 100-200 μl of cell staining buffer and analyze immediately in the cytometer, or alternatively cells can be resuspended in 2% paraformaldehyde (PFA) prepared in PBS and stored over-night at 4°C.
Representative Results:
After spinning the gradients, the 70%-30% interphase should have a defined white halo, and quantitation of the cells recovered from a naíve mouse should yield 3-5 x105 cells per mouse (brain plus spinal cord). Inflamed brain might have ranging numbers of inflammatory cells depending of the CNS insult or disease model (Figure 1).
<img src="/files/ftp_upload/2348/2348fig1.jpg" alt="Flow cytometry analysis; naïve vs EAE brain cells; scatter plots; CD45 leukocytes, microglia, differentiation." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
 Figure 1. Isolation of mononuclear cells, from naíve and EAE-brain at peak disease. Brain cells suspensions were separated over discontinuous 70%/30% percoll gradients. Cells stained were incubated with Fc-block followed by anti-CD45 antibodies for 30 min on ice. Cells were rinsed in cell staining buffer, and fixed in 2% PFA prior acquisition in an LSR-II. CD45 intensity is measured in the Y-axis, where three distinct populations are visualized. Further characterization of the immune phenotype of CD45hi infiltrating cells is implemented using additional antibodies and subsequently analyzed by flow cytometry.

<ol><li>Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+direct+characterization+of+resident+microglial+cells+from+the+normal+and+inflamed+central+nervous+system%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system.</a> Proceedings of the National Academy of Sciences of the United States of America. 88, 7438-7442 (1991).</li>
<li>Ponomarev, E. D., Shriver, L. P., Maresz, K., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglial+cell+activation+and+proliferation+precedes+the+onset+of+CNS+autoimmunity%5BTitle%5D)+AND+%22J.+Neurosci.+Res%22%5BJournal%5D)">Microglial cell activation and proliferation precedes the onset of CNS autoimmunity.</a> J. Neurosci. Res. 81, 374-389 (2005).</li>
<li>Ford, A. L., Goodsall, A. L., Hickey, W. F., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normal+adult+ramified+microglia+separated+from+other+central+nervous+system+macrophages+by+flow+cytometric+sorting.+Phenotypic+differences+defined+and+direct+ex+vivo+antigen+presentation+to+myelin+basic+protein-reactive+CD4%2B+T+cells+compared%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared.</a> J Immunol. 154, 4309-4321 (1995).</li>
<li>Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+murine+microglial+cells+for+RNA+analysis+or+flow+cytometry%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Isolation of murine microglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li>
<li>Juedes, A. E., Ruddle, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resident+and+infiltrating+central+nervous+system+APCs+regulate+the+emergence+and+resolution+of+experimental+autoimmune+encephalomyelitis%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Resident and infiltrating central nervous system APCs regulate the emergence and resolution of experimental autoimmune encephalomyelitis.</a> J. Immunol. 166, 5168-5175 (2001).</li>
<li>Prinz, M., Priller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tickets+to+the+brain%3A+Role+of+CCR2+and+CX%283%29CR1+in+myeloid+cell+entry+in+the+CNS%5BTitle%5D)+AND+%22J+Neuroimmunol%22%5BJournal%5D)">Tickets to the brain: Role of CCR2 and CX(3)CR1 in myeloid cell entry in the CNS.</a> J Neuroimmunol. , (2010).</li>
<li>Mildner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+in+the+adult+brain+arise+from+Ly-6ChiCCR2%2B+monocytes+only+under+defined+host+conditions%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions.</a> Nat. Neurosci. 10, 1544-1553 (2007).</li>
<li>Djukic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+monocytes+engraft+in+the+brain%2C+differentiate+into+microglia+and+contribute+to+the+pathology+following+meningitis+in+mice%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">Circulating monocytes engraft in the brain, differentiate into microglia and contribute to the pathology following meningitis in mice.</a> Brain. 129, 2394-2403 (2006).</li>
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No conflicts of interest declared.

Analyses of cell surface markers in CNS leukocytes from normal and inflamed mouse tissues has been utilized for several decades1-3. Isolation protocols are based in the separation of microglia and leukocytes by density centrifugation4. Here we describe a fast and effective method of isolating CNS leukocytes using discontinuous percoll gradients. After isolation of cells, staining with various antibodies such as -but not limited to- CD45, CD4, CD8, CD11b, CD19, etc. allows the identification of immun...

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Tissue homogenization and Gradients&lt;/p&gt;&lt;ul&gt;&lt;li&gt;Percoll (Amersham)&lt;/li&gt;&lt;li&gt;10X Hanks&amp;rsquo; Balanced Salt Solution, without Calcium and Magnesium (Gibco)&lt;/li&gt;&lt;li&gt;RPMI Medium 1640, without phenol red (Gibco)&lt;/li&gt;&lt;li&gt;7-ml or 15-ml Dounce Tissue Grinders (VWR)&lt;/li&gt;&lt;/ul&gt;&lt;p class=&quot;jove_step&quot;&gt;Flow cytometry&lt;/p&gt;&lt;ul&gt;&lt;li&gt;Cell Staining Buffer (Biolegend)&lt;/li&gt;&lt;li&gt;Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block), BD Pharmingen&lt;/li&gt;&lt;li&gt;Hemacytometer (Fisher)&lt;/li&gt;&lt;li&gt;Anti-mouse CD45 (clone 30-F11), CD4 (clone RM4-5), CD19 (clone 6D5), BioLegend&lt;/li&gt;&lt;li&gt;10X Phosphate Buffered Saline, without Calcium and Magnesium (Thermo Scientific)&lt;/li&gt;&lt;li&gt;Paraformaldehyde (Sigma Aldrich)&lt;/li&gt;&lt;li&gt;Flow cytometry analysis performed with a LSR II (BD Biosciences)&lt;/li&gt;&lt;/ul&gt;</td></tr></tbody></table>

isolation of brain and spinal cord mononuclear cells using percoll gradients

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 ...

Immune cells infiltrate the central nervous system during infection, trauma, autoimmunity, or neurodegeneration. While histochemical approaches can be used to determine the location of the infiltrating cells and to analyze the associated pathology, these techniques are limited by the number of antibodies that can be used simultaneously. Here a method for rapid isolation of mononuclear cells from brain and spinal cord tissues is shown in which the dissected tissue is first homogenized.Next, a perol gradient is set up and following centrifugation, the mononuclear cells are collected from the interface. Finally, the cells are rinsed and stained with antibodies Through flow cytometry analysis, hematogenous cells and surveillance microglia are distinguished and their relative proportions during disease states can be determined. The main advantage of this technique over existing methods like Aris gradient or in such a histochemistry, is that it is less time consuming.This allows you to process more tissues at the same time. Demonstrating the gradient setup will be palino A technician from my lab Prepare the reagents that will be needed for this protocol. Starting with four milliliters of stock isotonic perol referred to as SIP per brain.To do this, combine nine parts of perol with one part 10 times HBSS without calcium and magnesium mixed by inversion. Next, prepare 70%per call while mixing the prepared SIP with one times HBSS without calcium and magnesium. Then dispense two milliliters of the 70%per cole into a 15 milliliter polypropylene conical tube to collect tissue.For this procedure, place the dissected brains and spinal cords of all the study mice in beakers containing 10 milliliters of RPMI over ice. Next, transfer the tissue to a seven or 15 milliliter down homogenizer containing three milliliters of one times HBSS with a loose fitting size. A pestle gently make a cell suspension, then switch to a tight fitting size B pestle, and further dissociate the tissues.Once the tissue is homogenized, add RPMI or one times HBSS to a final volume of seven milliliters and proceed to setting up the gradient to set up the percoco gradient. Begin by adding three milliliters of SIP to the cell suspension to a final PERCOCO concentration of 30%Keep in mind that the perol should be used at room temperature. If it is used cold, the cells tend to clump and cell separation is less efficient.Next, using our pipette aid setting gravity mode, slowly layer the 10 milliliter cell suspension on top of the 70%SIP. Avoid mixing the 70%and 30%solutions. This is the most critical step of the procedure.A very clear flat line should be visible at the 70 to 30%junction. Generally, individuals new to this method will struggle because the third and 70%solutions will mix during the layering. Next step, compromising the interface Centrifuge 30 minutes of 500 Gs at 18 degrees Celsius.Make sure the centrifuge will stop with minimal or no break so that the interface is not disturbed. After spinning the gradients, a thick and viscous layer of debris should accumulate at the top of the tube. The next layer is the yellowish 30%per core layer, which sometimes contains debris.Next is the 70 to 30 interface, which should have a defined white halo and contains the mononuclear cells. Finally, at the bottom of the tube is the clear 70%perol layer. If no interface is observed, the yield of cells will likely be very low, making further analysis extremely difficult.Therefore, at that point, fresh cell suspensions might be required to decrease contamination. Carefully remove the layer of debris from the top of the tube and discard it. Then using a plastic pipette, go through the top 30%per core layer and collect two to three milliliters of the 70 30 interphase and transfer it to a clean conical tube containing eight milliliters of one times HBSS.Mix the diluted interfaces a few times by inversion, then centrifuge for seven minutes at 500 GS at 18 degrees Celsius. Following the spin, remove the supena and resuspend the pellet. In one milliliter of cell staining buffer, transfer the cell suspension to a 1.5 milliliter tube and wash it one more time.Using a micro centrifuge at 10, 000 GS for one minute at four degrees Celsius before proceeding with antibody staining to perform antibody staining, resuspend the pellets in 50 microliters of purified anti-US CD 16, CD 32, diluted one to 200 in cell staining buffer to block the FC binding sites. As for any staining procedure, it is imperative that all antibodies are adequately titrated incubate on ice for 10 minutes. During the incubation, remove two to five microliters of cells and count them using a hemo cytometer.The combined naive brain and spinal cord tissues should yield around three to five times 10 to the five cells per mouse. Inflamed brains will have ranging numbers of inflammatory cells depending on the time point at which they are collected and the disease model Following the incubation with CD 16 and CD 32, add 50 microliters of immune cell specific antibody cocktail mix. The mix used in this example contains antibodies against CD 45, CD four, and CD 19 incubate on ice for 30 minutes.Importantly, each antibody must be first titrated. To assess optimal dilution, use appropriate fluorochrome combinations chosen according to the capabilities of laser and filter settings of your particular flow. Cytometer, after staining the cells, wash them in cell staining buffer by adding one milliliter of cell staining buffer and spinning them for one minute.At 10, 000 Gs following the spin, discard the supine agent, then resuspend pallets in 100 to 200 microliters of cell staining buffer for immediate flow cytometric analysis for later analysis. Reus suspend the cells in 2%par formaldehyde prepared in PBS and store overnight at four degrees Celsius. Shown here are mononuclear cells obtained and incubated with FC block, followed by anti CD 45, CD four, and CD 19 antibodies.In accordance with the demonstrated protocol by flow cytometry CD 45 staining nicely separates CD 45 high hematogenous cells from CD 45, DIM surveillance microglia. This figure compares the cells isolated from naive and an inflamed brain from a mouse induced to develop experimental autoimmune encephalomyelitis. CD 45 A PC intensity is shown on the Y axis where three distinct populations are visualized in the naive brain.Very few numbers of CD 45 high cells are detected. Usually less than 2%of the total isolated population. In comparison brains from EAE affected mice show a massive influx of blood cells recruited to the brain.Addition of other cell surface markers such as CD 11 b, CD four, CD eight, et cetera, allows further characterization of the CD 45 high population Once mastered, this technique can be done in three to four hours if it is performed properly. While attending this procedure, it is important to remember that histological approaches in conjunction with immune phenotyping by flow cytometry are critical to fully characterize the composition of the CNS inflammatory infiltrates.

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Learn to isolate mononuclear cells from mouse brain and spinal cord using discontinuous Percoll gradients. This rapid protocol enables flow cytometric phenotyping of CNS-infiltrating immune populations.

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

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