microglia from the adult mouse spinal cord

Journal Pre-proof Isolation and RNA purification of macrophages/microglia from the adult mouse spinal cord

Ourania Tsatas, Nader Ghasemlou PII:

S0022-1759(19)30343-6

DOI:

https://doi.org/10.1016/j.jim.2019.112678

Reference:

JIM 112678

To appear in:

Journal of Immunological Methods

Received date:

18 August 2019

Revised date:

25 September 2019

Accepted date:

3 October 2019

Please cite this article as: O. Tsatas and N. Ghasemlou, Isolation and RNA purification of macrophages/microglia from the adult mouse spinal cord, Journal of Immunological Methods (2018), https://doi.org/10.1016/j.jim.2019.112678

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© 2018 Published by Elsevier.

Journal Pre-proof

Isolation and RNA purification of macrophages/microglia from the adult mouse spinal cord Ourania Tsatas1 and Nader Ghasemlou2,3,4* Centre for Research in Neuroscience, Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada; Departments of 2Biomedical & Molecular Sciences and 3 Anesthesiology & Perioperative Medicine; and 4Centre for Neuroscience Studies, Queen’s University, Kingston, Ontario, Canada

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*Corresponding author Address: Queen’s University, 18 Stuart St., room 754 Kingston, Ontario, K7L 3N6, Canada Phone: 613.533.6854 Fax: 613.533.2022 Email: [email protected]

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Type of article: Technical Note Declarations of interest: None.

Running title: Isolation and RNA purification of CNS macrophages Words: total = 2,345 Figures: 2; Tables: 0

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Journal Pre-proof Abstract The isolation of a highly pure and healthy population of macrophages from the CNS in a short time is essential for studying the role of these cells in injury and disease. Current methods rely either on the use of gradients and/or sorting, processes that result either in impure, reduced viability and/or altered populations of cells. Furthermore, RNA extraction after immunopanning is often difficult. Here, a technique combining both gradient isolation and immunopanning to

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generate highly pure cultures of primary macrophages isolated from the injured adult CNS in less

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than 2 hours is described. An optimized protocol of a commercially-available RNA extraction kit

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is also outlined for the isolation of highly pure RNA. A hybridoma cell-line producing CD11b

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antibody was used to recover activated (CD11b+) macrophages/microglia from the CNS after

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injury in less than 2hours with >95% purity.

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Keywords: macrophage; monocyte; microglia; spinal cord injury; cell purification;

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1. Introduction

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immunopanning; RNA extraction

The method first describing immunopanning, or the immuno-selective method to purify cells in vitro, was first published in 1975 for the specific isolation of T and B lymphocytes from human peripheral blood (Barker et al., 1975). The technique has since been used extensively to isolate cells from the peripheral and central nervous systems, including oligodendrocyte precursor cells, astrocytes, spinal motor neurons, and dorsal root ganglia sensory neurons, among others (Barres, 2014). Immunopanning protocols for the purification of microglia/macrophages from the injured

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Journal Pre-proof nervous system, particularly for the extraction of highly pure RNA (e.g., for microarrays), have only recently been described (Collins and Bohlen, 2018; Bohlen et al., 2019). Standard methods to isolate microglia/macrophages from the CNS rely on the use of enzymatic digestion of the tissue and/or multiple Percoll gradients (Sedgwick et al., 1991; Cardona et al., 2006). This may result in cell activation and/or cleavage of cell-surface receptors (with enzymatic digestion) or loss of cells. Furthermore, gradient isolation is often used in

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conjunction with either flow cytometry or antibody-conjugated magnetic bead sorting to increase

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the purity of isolated cells. This combined approach, however, can take much longer to complete

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and result in reduced viability and/or altered activation states. Our own observations suggest that

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flow cytometry and magnetic sorting methods are best suited to those models where activated microglia/macrophages make up a large proportion of total CNS cells but are less suited for

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applications where they do not, such as after focal injuries or diseases.

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There is therefore a need for methods to quickly and efficiently isolate macrophages from the spinal cord, particularly in injury models where there may be only a small number of

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macrophages present relative to other cells. To this end, we have developed a protocol to isolate

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these cells from the spinal cord in less than two hours by combining the gradient separation of leukocytes from the CNS with a modified immunopanning protocol. Furthermore, we have developed a method to extract RNA from cells isolated from the CNS that can be used for methods requiring highly pure RNA samples. After testing various protocols, including TRIzol and kits from various vendors, we found that a protocol using the RNEasy Lipid Tissue Mini kit from Qiagen provided the best results.

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Journal Pre-proof 2. Materials and Methods 2.1 Protocol 1: Macrophage isolation and purification from the spinal cord 2.1.1 Animals and surgery All experiments were approved by the McGill University Animal Care Committee following the guidelines of the Canadian Council on Animal Care. Female C57BL/6 mice (Charles River Canada) between 8-12 weeks of age were anesthetized with ketamine:xylazine:acepromazine

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(50:5:1 mg/kg) and a partial laminectomy made using Mouse Laminectomy Forceps (Fine

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Science Tools [FST], Vancouver) at the 10th thoracic vertebral level. The mice were

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immobilized with modified serrated Adson forceps (FST) attached to the immediately adjacent

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vertebrae, and the contusion injury performed using the Infinite Horizons impactor device (Precision Scientific Instrumentation, Lexington, KY), as described previously (Ghasemlou et

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al., 2005).

2.1.2 CD11b antibody generation

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The monoclonal CD11b antibody is generated using the M1/70 hybridoma cell line (Springer et

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al., 1978) (ATCC; #TIB-128), grown in Dulbecco’s Modified Essential Medium (DMEM; e.g., Invitrogen #12491-015) supplemented with 20% fetal calf serum, 1% penicillin –Streptomycin and1% MEM Vitamin solution. Cells were grown until confluent, the media gently collected, centrifuged and the supernatant stored in 0.5-1ml aliquots at -80C until ready for use. This supernatant comprises the CD11b antibody used to isolate macrophages/activated microglia from the nervous system. While antibody concentrations were not measured here, confluent cell supernatants from this hybridoma line have reported antibody concentrations of ~110μg/ml when grown to confluency (Ault and Springer, 1981). Antibody concentrations cannot be measured

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Journal Pre-proof using standard protein assays (e.g., Bradford, Lowry methods) due to the presence of fetal calf serum in the supernatant. Instead, enzyme-linked immunosorbent assay (ELISA) will have to be used to ensure equal loading for each batch used. Commercially-available antibodies are often provided at concentrations of ~1mg/ml, and should therefore be diluted (e.g., 1:10 in DMEM) if used; this could provide a cost-effective strategy to use this methodology without the need to

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maintain a hybridoma cell line.

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2.1.3 Preparation of antibody-coated petri dish

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One day prior to collection of cells, 500μl to 1ml of CD11b antibody is added to a 20-30mm

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Petri dish for at least 12h (preferably overnight) at 4C. Petri dishes are covered with parafilm to avoid evaporation and loss of antibody. It is best to use undiluted CD11b antibody obtained from

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confluent hybridoma cells. Diluting the antibody supernatant can result in lower purity of

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antibody due to variability.

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macrophages using the protocol outlined here. However, we recommend testing each batch of

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2.1.4 Preparation of Percoll gradient and tissue processing On the day of cell isolation, place a 70μm nylon mesh cell strainer (e.g., Corning #352350) onto the top of a 50ml falcon tube and wet it with 5ml of Minimum Essential Medium (MEM), supplemented with 25mM HEPES and glutamine (e.g., Invitrogen #42360-032). Prepare 80% and 40% Percoll from iso-osmotic Percoll solution (e.g, GE Life Sciences #17-0891-01). Keep all solutions and samples on ice or at 4C unless otherwise noted. Mice are sacrificed by guillotine or CO2, and the body sprayed with 70% ethanol. An incision is made along the back and the vertebral laminae exposed to the spinal cord. Using a scalpel or blade, the section of the

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Journal Pre-proof spinal cord to be removed for macrophage purification is excised and placed in a petri dish in MEM-HEPES. The spinal cord is then transferred onto the nylon cell strainer and the tissue minced into small pieces using micro-scissors. For isolation of macrophages from the brain, the tissue will need to be chopped into small pieces of about 5mm3, and passed through at least 3-4 separate 70μm nylon meshes per brain. Each sample run through a mesh should be immunopanned in a separate plate.

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Tissue dissociation can be done using various methods. While the use of a glass Dounce

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homogenizer is recommended as others have done for similar macrophage purification from the

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spinal cord (Greenhalgh et al., 2018), we used the soft end of the plunger from a 1ml tuberculin

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syringe to slowly pass the tissue through the mesh, using a circular motion while adding more MEM-HEPES as needed. The cells are then centrifuged at 600g for 15 minutes at 4C, the

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supernatant discarded, and the cell pellet resuspended in 3.5ml of the 80% Percoll in a 15ml

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conical tube. Slowly, 4ml of the 40% Percoll is overlayed and the conical centrifuged at 500g for 35 minutes at 4C. The layer of cells at the interface between the 80% and 40% Percoll solutions

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contains leukocytes. Using a fire polished glass pipette, this layer is removed and resuspended in

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up to 10ml of Hank’s Balanced Salt Solution (HBSS; e.g, Invitrogen #14025-092) in a 15ml conical tube. The cells are centrifuged at 600g for 15 minutes at 4C, and the supernatant discarded. This cell pellet is then used for immunopanning.

2.1.5 Immunopanning to isolate macrophages The antibody-containing medium is aspirated from the 20mm dishes and left to sit uncovered for 2-3 minutes. The cell pellet is resuspended in 1.5ml of Advanced RPMI 1640 media (e.g., Invitrogen #12633020) supplemented with 10% fetal bovine serum (e.g., Invitrogen #16000-

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Journal Pre-proof 036), 1% penicillin/streptomycin (e.g., Invitrogen #15070063) and 1% MEM vitamin solution (e.g., Invitrogen #11120052). For optimal results, cells from a 5mm segment of spinal cord are resuspended in 1.5ml of medium, to ensure proper binding of cells to the antibody-coated plates. Up to 1.5ml of cell suspension is then added to the Petri dish and incubated at 37C for 10 minutes. The dish is gently swirled and incubated for another 11 minutes (for a total incubation time of 21 minutes). The noted times are specific to the batch of hybridoma cells used in our

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laboratory and should be adjusted in each laboratory. Once incubation is completed, the dish is

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gently washed with RPMI at room temperature to remove unbound floating cells, and the

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supernatant discarded. Bound cells are gently triturated by adding 3 x 1ml RPMI using a fire

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polished pipette. Dissociated cells are transferred to a 15ml Falcon tube and centrifuged at 600g for 15 minutes at room temperature, the supernatant aspirated and cell pellet used in further

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experiments.

2.2 Protocol 2: RNA extraction from isolated macrophages

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The Qiagen RNEasy Lipid Tissue Mini kit (Qiagen #74204) was used to extract RNA from

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purified cells. After aspirating the supernatant from the cell pellet, 1ml QIAzol lysis reagent (Qiagen #79306) is added to the cell pellet and the cells resuspended in lysis solution with gentle pipetting. The QIAzol solution containing lysed cells is transferred to a 2ml Eppendorf tube and vortexed to ensure cells are completely lysed with the tube placed onto a rocker for 5min at room temperature. 200μl chloroform (e.g., Sigma Aldrich #319988) is added and the tube shaken vigorously for 15sec, and the tube left undisturbed at room temperature for 3 minutes. Following centrifugation at 12,000g for 15 minutes at 4C, the upper aqueous phase is transferred to a new 1.5ml Eppendorf tube. From this point onwards, all steps are carried out at room temperature

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Journal Pre-proof unless otherwise noted. 1 volume of 70% ethanol is added to the tube and mixed by vortexing. This solution is pipetted onto the supplied Qiagen Mini Spin column and centrifuged. 500μl of Buffer RW1 is added and left on the column for 3min, and the column then centrifuged at 9,000g for 15 sec. The collection tube (supplied by manufacturer) is changed and 500μl Buffer RPE added and left on the column for 3min, followed by centrifugation at 9,000g for 15 sec. 500μl 80% ethanol is added and left on the column for 3min followed by centrifugation at 9,000g for

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2min. The collection tube is changed again and the column spun dry for 5min at 9,000g with the

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cap open. RNase-free water warmed to 37C is added to the center of the column and left for

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4min, followed by centrifugation at 9,000g for 1min. The resulting RNA sample can then be

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assessed using a BioAnalyzer or other method to measure RNA quality.

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3. Results and Discussion

Our protocol for the immunopanning of macrophages from both microglial and monocytic origin

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provides one of the first described methods for the isolation of highly pure CD11b+ cells from

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the central nervous system. Microglia, which make up a large proportion of CNS glia, play an important role in both the normal and injured nervous system. After injury or disease, circulating blood monocytes can enter the CNS where they become activated. Our work shows that activated microglia and macrophages can be isolated from the central nervous system using this modified immunopanning technique in approximately 2 hours total time (see Figures 1 and 2). The final population obtained is > 95% CD11b+ cells, when isolating cells from the spinal cord after moderate contusion injury (Ghasemlou et al., 2005). Cells were quantified by measuring the percentage of CD11b immunopositive cells (using a rat anti-mouse antibody [Bio-Rad,

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Journal Pre-proof #MCA711]), relative to all DAPI-stained nuclei in at least 3 replicate dishes for each condition tested. We have found that incubation of longer than 21 minutes will result in a reduced purity of macrophages. For instance, a 30 minute incubation will result in a 75% CD11b+ cell purity, while a 21 minute incubation will yield 95% purity. Meanwhile, shorter incubation times result in too few cells recovered. The use of undiluted primary antibody obtained from the hybridoma cell line is preferred, as there was a reduced purity observed when the antibody used to coat Petri

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dishes was diluted. Use of commercially-available antibody from the same M1/70 clone is

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expected to produce similar results, but should be diluted prior to use in DMEM or other buffer.

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Use of a Dounce glass homogenizer is also expected to increase overall yield and reproducibility,

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and is recommended. It is important to note that this protocol was optimized for use of spinal cord tissue, though we do not expect differences using brain tissue.

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Once activated, it is difficult to differentiate between microglial- and monocyte-derived

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macrophages, though specific markers have been identified potentially delineating the two populations (for example, Tmem119 or levels of CD45 expression) (Bennett et al., 2016). Thus,

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other antibodies may be used for immunopanning specific subsets of macrophages from the

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nervous system, by separating cells based on origin (e.g., Tmem119) or activation state (e.g., CCR2). The efficiency of cell isolation and purity of cells using antibodies other than CD11b is unknown and worthy of future study. Recent work using a similar immunopanning method was developed for the isolation of rat microglia from the brain under serum-free conditions (Collins and Bohlen, 2018), with low numbers of macrophages found in the culture. Thus, our two immunopanning methods could be used to compare and contrast activation states and marker expression of both quiescent and activated microglia. Better understanding the functions of isolated CNS macrophages/microglia, such as by cell culture or gene expression analysis using

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Journal Pre-proof RNASeq or microarray, will be important for deciphering their role in health and disease. While we have not optimized protocols using these isolated cells for in vitro work, others have used macrophages isolated from the spinal cord using similar protocols for up to 24 hours in culture (Greenhalgh et al., 2018). However, our focus use of these cells for RNA expression analysis. RNA of high purity and quality can also be extracted from cells isolated using our immunopanning method. However, our work suggests that standard techniques used to isolate

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this RNA may require adjustment and optimization when cells are isolated by immunopanning.

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We began our work by first testing three different commercially-available RNA isolation kits

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(TRIzol Plus RNA Purification kit from ThermoFisher; and the RNeasy and RNeasy Lipid

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Tissue Mini kits from Qiagen). All commercially-available kits were used encountered several issues, including poor RNA extraction, excessive DNA contamination (often even with use of

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on-column DNase digestion), salt contamination, and sheared/degraded RNAs (see Figure 3).

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However, we found that the Qiagen RNeasy Lipid Tissue Mini kit provided the highest yield, with similar purity, of the three kits; our subsequent work focused on optimizing this protocol

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from that provided by the manufacturer. All changes made to this protocol are outlined here.

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Firstly, in an effort to increase yield and reduce cell manipulation, we added lysis solution directly to the Petri dishes where cells were purified. Small cellular fragments were occasionally seen in the QIAzol lysis solution during this step, possibly due to neutralization of the lysis solution by the antibody coating the plate. This was avoided by doubling the volume of lysis reagent used to 2ml. Secondly, the buffers used in the kit (RW1 and RPE) are allowed to incubate on the RNA columns for a minimum of 2min before each wash step. This resulted in increased purity of RNA. Finally, the RNase-free water used to extract RNA from columns should be heated to 37C and amounts adjusted based on expected yield, with no less than 14μl

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Journal Pre-proof applied. Furthermore, the RNase-free water should be left on the columns for 1-2min prior to centrifugation to increase yield. If concentrated RNA is required, the eluted RNA may be reapplied to the column and re-centrifuged. There will, however, be a lower volume of total RNA with this procedure. If high RNA yield is expected, as with samples with larger starting material, 14-20μl of warm RNase-free water may be added twice (for a total of 28-40μl of total RNA), which can then be concentrated further, if necessary, using concentration kits (such as Qiagen

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RNEasy MinElute Clean Up kit; Qiagen #74804).

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To conclude, the macrophage isolation method outlined here is cost-effective,

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reproducible, and in contrast to existing protocols, such as magnetic cell sorting and flow

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cytometry, requires only standard laboratory equipment. Our optimized RNA extraction protocol

Acknowledgements

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for use with cultured cells as well.

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also allows for a relatively pure RNA samples from these isolated cells, and may hold promise

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This work was supported by grants from the Bryon Riesch Paralysis Foundation, the Canadian

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Pain Society/Pfizer Canada, and Canadian Institutes of Health Research (to NG). The authors thank Dr. Samuel David for scientific contributions and editing a first draft of the manuscript.

Declarations of interest: None.

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Journal Pre-proof 4. References

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Ault, K.A. and Springer, T.A., 1981, Cross-reaction of a rat-anti-mouse phagocyte-specific monoclonal antibody (anti-Mac-1) with human monocytes and natural killer cells. J Immunol 126, 359-64. Barker, C.R., Worman, C.P. and Smith, J.L., 1975, Purification and quantification of T and B lymphocytes by an affinity method. Immunology 29, 765-77. Barres, B.A., 2014, Designing and troubleshooting immunopanning protocols for purifying neural cells. Cold Spring Harb Protoc 2014, 1342-7. Bennett, M.L., Bennett, F.C., Liddelow, S.A., Ajami, B., Zamanian, J.L., Fernhoff, N.B., Mulinyawe, S.B., Bohlen, C.J., Adil, A., Tucker, A., Weissman, I.L., Chang, E.F., Li, G., Grant, G.A., Hayden Gephart, M.G. and Barres, B.A., 2016, New tools for studying microglia in the mouse and human CNS. Proceedings of the National Academy of Sciences of the United States of America 113, E1738-46. Bohlen, C.J., Bennett, F.C. and Bennett, M.L., 2019, Isolation and Culture of Microglia. Curr Protoc Immunol 125, e70. Cardona, A.E., Huang, D., Sasse, M.E. and Ransohoff, R.M., 2006, Isolation of murine microglial cells for RNA analysis or flow cytometry. Nat Protoc 1, 1947-51. Collins, H.Y. and Bohlen, C.J., 2018, Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium. J Vis Exp. Ghasemlou, N., Kerr, B.J. and David, S., 2005, Tissue displacement and impact force are important contributors to outcome after spinal cord contusion injury. Experimental neurology 196, 9-17. Greenhalgh, A.D., Zarruk, J.G., Healy, L.M., Baskar Jesudasan, S.J., Jhelum, P., Salmon, C.K., Formanek, A., Russo, M.V., Antel, J.P., McGavern, D.B., McColl, B.W. and David, S., 2018, Peripherally derived macrophages modulate microglial function to reduce inflammation after CNS injury. PLoS Biol 16, e2005264. Sedgwick, J.D., Schwender, S., Imrich, H., Dorries, R., Butcher, G.W. and ter Meulen, V., 1991, Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system. Proceedings of the National Academy of Sciences of the United States of America 88, 7438-42. Springer, T., Galfre, G., Secher, D.S. and Milstein, C., 1978, Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens. Eur J Immunol 8, 539-51.

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Journal Pre-proof 5.

Figure Legends

Figure 1. Procedure workflow outlining the different steps for isolation and purification of microglia/macrophages from the CNS (Protocol 1). Expected percent total macrophages are based on work carried out using spinal cords from C57BL/6 mice 7 days after moderate SCI using the Infinite Horizons spinal cord impactor.

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Figure 2. Flow chart describing key steps for (A) macrophage/microglial isolation from the

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nervous systems and (B) RNA extraction of cells.

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Figure 3. Agilent BioAnalyzer 2100 readouts showing RNA quality. The first peak at 22s corresponds to the marker, and the peaks at 42 and 48s correspond to the 18S and 28S RNAs. A

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peak for 5S RNA is occasionally seen immediately after the marker peak (as in C). (A-C)

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Specific problem areas include low abundance of RNA, (i) salt contamination, (ii) genomic DNA contamination, and (iii) sheared and/or degraded RNAs. Salt contamination often appears as

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peaks after the marker and can alter the run time in seconds, changing the location of the 18S and

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28S RNA peaks. (D) A BioAnalyzer readout showing RNA of high quality, with no sheared/degraded RNA, genomic DNA or salt contamination, is acceptable for use in molecular biology assays, such as microarrays.

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Journal Pre-proof Highlights 

Immunopanning with a hybridoma-derived CD11b antibody is used to isolate macrophages and activated microglia from the central nervous system

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Cell isolation requires no new laboratory equipment, is cost-efficient, results in a highly pure population of cells, and takes less than 2 hours to complete Commercially-available RNA extraction kits have been optimized to obtain RNA suitable

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for molecular biology analysis

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Figure 1

Figure 2

Figure 3