Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Summary

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

Abstract

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson's, Alzheimer's, and Huntington's Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Introduction

Microglia are Myb-independent resident macrophages of mesodermal origin, which differentiate from c-kit+/CD45- erythromyeloid progenitors in the blood islands of the yolk sac^1,2. Once embryological microglia have colonized the central nervous system (CNS), they transition from an amoeboid to a ramified form³. These adult microglia are classified as surveillant since their dynamic ramifications probe the healthy brain parenchyma for potential insults⁴. Although microglia only contribute to approximately 10% of the CNS cell population, their ability to tile amongst each other ensures maximal scanning of the parenchyma^4,5. Danger-associated molecular patterns (DAMPs), such as α-synuclein^6,7 and amyloid-β⁸, or pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS)⁹, classically activate microglia to promote an inflammatory response characterized by reversion to the amoeboid active state and the production of nitric oxide, tumor necrosis factor-α (TNFα), interleukin 1β (IL-1β), IL-6, IL-12, and the chemokine C-C motif ligand 2^9,10,11. In neuroinflammatory conditions such as Parkinson's disease, in which pathogenic α-synuclein has accumulated, a neurodegenerative cycle is created from the death of dopaminergic neurons, which release more aggregated α-synuclein, further promoting classical activation of microglia⁷. Similar to peripheral macrophages, microglia may also have the ability to alternatively activate in the presence of the anti-inflammatory cytokines IL-4 and IL-10, giving them the potential of promoting neural repair and attenuating inflammation^2,11. Aside from their immunological roles in the CNS, microglia have been described as vital regulators of neuronal circuitry by pruning synapses during development. For example, Cx3cr1-KO mice have less dense microglia and reduced synaptic pruning, which leads to an overabundance of dendritic spines, immature synapses, and the electrophysiological patterns of an underdeveloped CNS¹². Understanding these physiological complexities and the diverse functional roles of microglia in the homeostasis of the CNS is critical to the search for therapeutics targeting neurodegenerative disorders.

In the area of neuroimmunology, in vitro experiments are highly desirable because of the greater feasibility for mechanistic studies, the lower maintenance costs, and for being less time- and labor-intensive. Furthermore, the ability to isolate cell populations is critical to delineate the functionality of those target cells under prescribed conditions. Numerous microglial isolation methods exist, but they are limited by their ability to obtain relatively high numbers and purity for broad experimentation^13,14,15. For example, a cluster of differentiation 11b (CD11b) is a common surface marker of monocytes, macrophages, and microglia¹⁶. By exploiting CD11b, a method of magnetic separation was first described as a column-based approach that yielded ~99.5% purity and ~1.6 x 10⁶ microglia per neonatal brain¹⁷. Our laboratory recently developed a column-free CD11b magnetic separation method¹⁵, which we performed in a polystyrene tube by tagging CD11b with a monoclonal antibody conjugated to phycoerythrin (PE). A bispecific secondary antibody to PE and dextran complexes with the PE. Once bound, dextran-coated magnetic particles are introduced, which bind to the dextran end of the antibody complex. Lastly, the polystyrene tube is placed in a magnet for microglial isolation. This approach doubled the yield to ~3.2 x 10⁶ microglia per neonatal brain but at the cost of reducing purity to ~97%.

Herein, we demonstrate a rapid and refined column-free CD11b magnetic separation protocol (Figure 1). This improved method remains as feasible as our original column-free method since the price of the CD11b magnetic separation kit is the same. The completion time is reduced in half, which can be crucial to maximizing cell survival and yield. Notably, the purity achieved from this optimized method is ~>99%, a marked improvement over the purity achieved from the original column-free method developed by our laboratory¹⁵. Most importantly, CD11b-PE is not utilized, eliminating the need to incubate away from light and allowing the use of the red channel for fluorescence microscopy. Lastly, as in the original CD11b method, an astrocytic fraction of high yield and purity is obtained with this improved method. Astrocytes are the most numerous glial cells in the CNS, leading to the idea that their homeostatic functions are indispensable in relation to pathophysiology¹⁸. These glial cells play a role in diverse physiological functions such as forming the blood-brain barrier, providing nutrient support, maintaining neurotransmitter homeostasis, forming glial scars in response to injury, neuroprotection, learning and memory, and neuroinflammation, exemplifying their investigatory potential in glial biology¹⁹. Morphology and functionality of microglia and astrocytes have been ascertained via confocal microscopy, Western blotting, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), Griess nitrite assay, and the Luminex multiplex cytokine assay. The refinement provided by this protocol offers increased confidence pertaining to microglial or astrocytic purity, broader application of fluorescence microscopy with the availability of the red channel, and saves time, all of which are important for in vitro experimentation.

Protocol

Use of the animals and protocol procedures were approved and supervised by the Institutional Animal Care and Use Committee (IACUC) at Iowa State University (Ames, IA, USA)

1. Growing of Mixed Glial Cultures

1. Decapitate 1- 2 day-old pups quickly with 5.5 inch operating scissors, and place the heads immediately in a 50 mL tube on ice. Note that this decapitation is the mode of euthanasia.
2. In a laminar airflow hood, make a small incision in the skull and meninges using 4.5 inch straight micro-dissecting scissors. Begin cutting from the caudal end to the rostral end (nose). Get underneath the skin by using the opening formed by the decapitation.
   1. After the incision, peel one of the hemispheres to the side. Then use a pair of curved or hooked tweezers to remove the entire brain.
3. Immerse the brain(s) in a new 50 mL tube containing 0.25% trypsin-ethylenediaminetetraacetate acid (EDTA) for 15 min in a 37 °C water bath. Use 2 mL of trypsin-EDTA per brain.
4. Wash the brain(s) with fresh Growth Media (10% FBS, DMEM/F12, 1% penicillin/streptomycin, 1% L-glutamine, 1% sodium pyruvate, and 1% non-essential amino acids) by adding and removing the media. Repeat this 4x.
5. For each brain, plate two T-75 flasks containing growth media. Therefore, add 2 mL of growth media per brain to the tube. Thus, it will be an equivalent of 1 mL of homogenized brain with 8-9 mL of growth media per T-75 flask.
6. Homogenize by triturating the brain(s) with pipettes of differing aperture sizes, in order from largest to smallest. When it is visible that the brain tissue is not getting smaller, transition to the next pipette. At the end of the trituration, the suspension should be clear, with no visible chunks.
   1. Use a 25 mL pipette, 5 mL pipette, and then a 10 mL pipette sequentially.
7. Pass each homogenous brain suspension through a 70 µm cell strainer to make it into a single cell culture.
8. For each homogenized brain, plate two T-75 flasks containing growth media, as described in step 1.5 (1 mL of homogenized brain with 8-9 mL of growth media per T-75 flask).
9. Change growth media after 6 d and grow until isolation on the 16^th day.

2. Isolation of Microglial Cells

1. After 16 days, remove the growth media from the flask and place it in a fresh 50 mL tube. Add 3 mL 0.25% trypsin-EDTA to each T-75 flask. Shake the flasks for 5 min at RT on an orbital shaker.
   1. Centrifuge the removed growth media at 0.4 x g for 5 min and use to stop the trypsin-EDTA reaction in the subsequent step.
2. After shaking for 5 min, add a minimum of 4 mL of growth media (fresh or the used media from 2.1.1) to stop the trypsin-EDTA reaction.
3. Triturate to ensure that all the cells have been detached.
4. After trituration, pass the cells through a 70 μm cell strainer to make it into a single cell culture, perform a cell count, and then spin down the cells at 0.4 x g for 5 min.
5. For every 100 x 10⁶ cells (roughly 15 x T-75 flasks), use 1 mL of Recommended Media (2% FBS, DPBS (calcium and magnesium chloride-free), 1 mM EDTA) to resuspend the cell pellet.
   NOTE: All following steps are tailored for a 1 mL separation.
6. Take a 5 mL polystyrene tube, add 1 mL of Recommended Media, and mark the meniscus. Add Recommended Media up to 2.5 mL and mark this as well. Discard the Recommended Media and transfer the re-suspended cells to the 5 mL polystyrene tube.
7. Add 50 µL of rat serum for every 1 mL of suspended cells. Incubate for 5 min at RT.
8. Prepare selection cocktail by mixing 25 µL of component A and 25 µL of component B. These components are proprietary.
9. Add 50 µL of the selection cocktail to the cells. Incubate for 5 min at RT.
   NOTE: For purer cultures, repeat step 2.9 (recommended but not mandatory).
10. Vortex microspheres for 45 s. Add 80 µL of microspheres per 1 mL of sample. Incubate for 3 min at RT.
11. Bring the volume to 2.5 mL in the polystyrene tube by adding the Recommended Media.
12. Put the tube in the magnet for 3 min at room temperature. Adjust the incubation to increase purity. Slowly pour out the Recommended Media into a 15 mL tube with the magnet still in the polystyrene tube.
13. Repeat step 2.12 three more times. Additional magnetic incubations may be performed to increase purity.
14. Add 3 mL of growth media and count the number of cells using a cell counter.
15. Plate cells accordingly in Poly-D-Lysine (PDL)-coated plates for treatments. Treat the cells 48 h after seeding inPDL-coated plates. This allows the cells to recover from the stress of separation.
    NOTE: Check the purity of the culture using immunocytochemistry as described previously¹⁵.
16. Plate the negative fraction (collected in a 15 mL tube), which mostly contains astrocytes, in T-75 flasks in the growth medium.
17. After at least 6 h of incubation in 37 °C incubator, change the medium and let it grow O/N. Split the astrocytes the next day for treatments.

Results

Microglia isolated using CD11b Positive Selection kit II have high purity

Primary mouse microglia were isolated using the above mentioned protocol and plated on poly-D-lysine-coated coverslips to check the purity of isolation. Ten thousand cells were plated per well and immunocytochemical analysis was performed using ionized calcium-binding adaptor molecule 1 (Iba1) as a marker of microglia and glial fibrillary acidic protein (GFAP...

Discussion

Older microglial isolation methods have limited recoveries that are not appropriate for various protein analyses by Western blot and RNA analyses by qRT-PCR. The differential adherence and mild trypsinization methods are two common approaches with low microglial yields^13,14,15. The column-based CD11b approach also has low recovery, but achieves greater purity than differential adherence and mild trypsinization^1...

Materials

Name	Company	Catalog Number	Comments
EasySep CD11b Separation Kit II	StemCell Technologies	18970
EasySep Magnet	StemCell Technologies	18000
DMEM/F12 (1:1) (1x)	Life Technologies	11330057
Sodium Pyruvate	Life Technologies	11360070
MEM Non-essential amino acids (100x)	Life Technologies	11140050
L-Glutamine (100x)	Life Technologies	25030081
EDTA	Fisher Scientific	AM9260G
Fetal Bovine Serum (FBS)	Sigma	13H469
0.25% Trypsin-EDTA	Gibco by Life Technologies	25200
Dulbecco's PBS (DPBS)	Gibco by Life Technologies	14190250