Novel In Vitro Culture of Microglia from Aged Mice: Implications for the Future of Aging Neurobiology Research

Abstract

Aging is associated with elevated levels of inflammation across tissues, a status recognized as “inflammaging.” Within the brain, microglia are the resident phagocytic immune cells that are important in both homeostatic and disease states. Aged microglia are susceptible to processes of “inflammaging,” which can include higher expression of baseline levels of inflammatory signals, decline in functional activity, and contribution to neurodegenerative processes. Information about microglial function has been gained using in vitro cell culture methods; however, most studies described previously have used microglia cultured from neonatal mice. More recent studies have used microglia cultured from young adult mice, but those using microglia from aged mice are lacking. Considering the distinct changes that come with aging and the important role of microglia in age‐related neurologic disorders, there is a need for reliable protocols for studying aged cells specifically. Here, we describe a method to culture primary microglia from aged mice. Collected brain tissue is digested using enzymatic and mechanical techniques and then cultured in specific medium that supports the continued survival and proliferation of adult and aged microglia. To confirm microglial identity, cultured cells were immunostained for microglia‐specific markers and imaged by microscopy and flow cytometry. We also compared the activation status of adult and aged microglia that were cultured versus those that were assessed directly after collection. Microglial cultures can easily be manipulated via genetic modifications or pharmacologic intervention to test specific functions. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Culturing primary microglia from adult and aged mice

INTRODUCTION

The central nervous system (CNS) is an immune‐regulated site that limits the entry of leukocytes and other peripheral immune cells. The CNS maintains its own population of tissue‐resident macrophages, known as microglia, which comprise 5% to 20% of the CNS glial cell population (Cronk & Kipnis, 2013; Gomez Perdiguero et al., 2013; Yang et al., 2010). Microglia provide critical functions in CNS growth and maintenance, including promoting cellular development, removal of myelin debris, and synaptic remodeling, all of which are required for CNS homeostasis (Cronk & Kipnis, 2013; Doorn et al., 2015; Vijaya et al., 2023). Microglia also participate in immunosurveillance of the brain parenchyma, surveying the environment for pathogens and injury (Cronk & Kipnis, 2013; Nimmerjahn et al., 2005; Yang et al., 2010). This occurs via extension of ramified processes from the cell somata, which engulf cellular debris and metabolic waste byproducts. Under homeostatic conditions, microglia continuously extend and retract new protrusions, permitting continual sampling of the local tissue microenvironment (Nimmerjahn et al., 2005).

Upon infection or tissue injury, microglia become activated, undergo rapid proliferation, and transition from a ramified to ameboid phenotype that promotes migration (Doorn et al., 2015; Qin et al., 2023; Saijo & Glass, 2011; Smith et al., 2012; Yang et al., 2010). This is accompanied by an increase in phagocytic ability; release of proinflammatory cytokines, including interleukin (IL)‐6, IL‐1β, and tumor necrosis factor (TNF); production of cytotoxic reactive oxygen and nitrogen species; and antigen presentation to elicit an adaptive immune response (Doorn et al., 2015; Qin et al., 2023; Smith et al., 2012; Saijo & Glass, 2011; Yang et al., 2010). This response stimulates the proliferation of other microglia and astrocytes within the brain and recruits peripheral lymphocytes for pathogen clearance and removal of tissue debris (Yang et al., 2010). Microglial activation can differ regionally (Doorn et al., 2015) and have different pathological outcomes, ranging from proinflammatory to immunoquiescent and neuroprotective. Recent studies indicate that although activated microglia have traditionally been categorized as strictly pro‐ or anti‐inflammatory, microglial activation in fact exists as a spectrum of gene expression profiles (Olah et al., 2020) and numerous morphological and transcriptomic characteristics (Gerrits et al., 2020; Salamanca et al., 2019). For example, whereas viral infection of the CNS and subsequent increase in interferon γ (IFNγ) lead to a proinflammatory profile, as described above (Smith et al., 2012; Yang et al., 2010), exposure to IL‐4 released by Th2 cells elicits a more anti‐inflammatory microglial response, with the secretion of factors such as IL‐10 and transforming growth factor β (TGFβ) (Qin et al., 2023), which can promote neurogenesis (Butovsky et al., 2006). However, this type of activation can still be maladaptive within some contexts, such as cancer (Saijo & Glass, 2011). These data are indicative of the complex nature of microglia, both at steady state and upon activation.

Advanced age is a major factor that affects microglial phenotype and function. Aged individuals have higher basal levels of inflammation, a condition termed “inflammaging,” which increases risk of many diseases, including cancer and neurodegeneration (Ferrucci & Fabbri, 2018). Aged microglia show differential gene transcription compared with the microglia of younger adult animals, expressing higher levels of activation markers, including CD45, CD68, and major histocompatibility complex (MHC) type II (Galatro et al., 2017; Godbout et al., 2005; Grabert et al., 2016). Aged microglia also show increased phagocytic ability and prolonged proliferation and activation, even after resolution of the inflammatory event (Antignano et al., 2023; Godbout et al., 2005; Luo et al., 2010; Miller & Streit, 2007; Xu et al., 2022). This “primed” characteristic of aged microglia promotes a rapid and robust inflammatory response; however, persistent microglial activation can disrupt homeostatic function and promote release of proinflammatory factors that can contribute to neurodegeneration and disease pathology (Cronk & Kipnis, 2013; Godbout et al., 2005; Smith et al., 2012; Saijo & Glass, 2011; Vijaya et al., 2023).

Microglia play a critical role in clearing damaged tissue debris and pathological protein aggregates. Immunoreactive microglia cluster around amyloid beta (Aβ), a pathological hallmark of Alzheimer's disease (Luo et al., 2010), creating a barrier to prevent neurotoxic spread (Condello et al., 2015). With age, microglial function declines, resulting in reduced efficacy of protein clearance and enhanced release of neurotoxic inflammatory mediators, in part due to decreased expression of CD36 and MMP9, which recognize and proteolyze Aβ fibrils, respectively (Floden & Combs, 2011; Hickman et al., 2008). Additionally, transcriptional profiles in aged microglia shift from neuroprotective to neurotoxic, which is strongly associated with loss of dopaminergic neurons in aged Parkinson's disease patients (Sawada et al., 2008). Thus, the contribution of microglia to neurodegenerative processes and whether they are protective or pathogenic, as well as how their contribution changes during aging, are areas of active investigation.

Insight into the phenotypic and functional changes that microglia undergo with advanced age will improve our understanding of age‐related dysfunction and microglial roles in neurodegenerative processes. Although prior studies have examined microglial activation and transcriptomic profiles using murine models of aging and neurological disease and post‐mortem patient samples (Gulyás et al., 2011; Keren‐Shaul et al., 2017; Olah et al., 2018; Streit et al., 2004), distinguishing the role of microglia on their own can be difficult due to the presence of other CNS‐resident cell populations or infiltrating peripheral cells. The use of in vitro cell culture provides a complementary tool to study the impact of microglia in a reductionist model. Conventional murine microglial culture methodologies utilize mixed glial cell cultures from young postnatal mice, followed by isolation or selection for microglia (Pesti et al., 2024). Use of neonatal mice maximizes initial microglial growth and polarization, which is required for successful in vitro culture; however, this method does not capture the aged microglial phenotype. Estimates indicate that mice aged 18 months correspond with human cohorts of ∼60 years of age based on survival rate and body composition (Jackson et al., 2017; Nagy & Pappas, 2019). Considering that Alzheimer's disease primarily affects individuals who are over the age of 65 (Alzheimer's Association, 2024), the use of mice that are ∼18 months of age is appropriate for research studying neurodegenerative processes. Currently, there are few published protocols that describe the isolation and culture of microglia from aged murine models (Vijaya et al., 2023). Here, we describe a methodology for the successful isolation and in vitro culture of microglia from 18‐month (mo)‐old and 8‐week (wk)‐old adult control C57BL/6J mice; these cells can be maintained in culture for up to 1 month.

NOTE: All procedures should be performed in compliance with appropriate institutional guidelines, such as Institutional Animal Care and Use Committee guidelines, and in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the International Guiding Principles for Biomedical Research Involving Animals.

CULTURING PRIMARY MICROGLIA FROM ADULT AND AGED MICE

The goal of this protocol is to obtain live microglia from adult and/or aged mice that can be cultured and grown in vitro. Although the culture process for microglia from aged mice is similar to that for microglia from adult mice, optimal culture conditions require specific growth media. If conducted properly, this procedure should yield ∼1 × 10⁶ viable cells per mouse brain (i.e., from two cortices). This number is sufficient for plating in two wells of a 12‐well plate (∼5 × 10⁵ cells per well). Cells typically reach maturity in ∼14 days and can be maintained in culture for ∼30 days. Usual precautions for handling animal cells in culture should be taken to maintain biosafety and sterility.

Materials

• Poly‐d‐lysine (PDL; Millipore Sigma, cat. no. P7280)

• 1× borate buffer (diluted from 20×; Thermo Fisher Scientific, cat. no. 28341)

• Dulbecco's phosphate‐buffered saline (dPBS), sterile (1×; Gibco, cat. no. 14190‐235), room temperature and 4°C

• Sterile water

• C57BL/6J mice (8 wk and 18 mo old)

• 70% (v/v) ethanol (EtOH; molecular biology grade, denatured; Fisher Bioreagents, cat. no. BP82031GAL)

• Trypsin‐EDTA (Trypsin 0.05%‐EDTA, phenol red, Gibco, cat. no. 25300‐054)

• Dissection tools:

• Pins

• Tissue forceps

• Surgical scissors

• Clips

• Dissection scoop (optional)

• Razor blade

• Autoclave

• Self‐sealing sterilization bags

• 150‐, 250‐, and 500‐ml vacuum filters (VWR, cat. no. 10040‐460, VWR, cat. no. 10040‐464, and Fisher, cat. no. FB12566510, respectively)

• Non‐tissue culture–treated 12‐well plates (Greiner Bio‐One, cat. no. 665102)

• 15‐ and 50‐ml conical tubes (Falcon, cat. no. 352097, and VWR, cat. no. 89039‐662, respectively)

• Isoflurane induction chamber

• Nose cone

• 20‐ml Luer‐Lok syringes (BD, cat. no. 302830)

• 20‐G hypodermic needles (BD, cat. no. 305175)

• Small tissue culture dishes (non‐treated, sterilized; VWR, cat. no. 10861‐594)

• Zip‐top bag with paper towel

• Orbital shaker or rocker

• 70‐µm cell strainers (Falcon, cat. no. 352350)

• 5‐ml syringe plungers (from 5‐ml Slip Tip syringes, BD, cat. no. 309647)

• Refrigerated centrifuge (capable of 500 × g and 1200 × g), 4°C

• Brightfield microscope

• Additional reagents and equipment for preparing microglia growth medium, microglia growth medium + GM‐CSF, isotonic Percoll, 37% isotonic Percoll, and collagenase digestion buffer (see recipes)

NOTE: It is important to use non‐tissue culture–treated plates that are then coated with PDL.

NOTE: All solutions and materials that come into contact with cells should be sterile. Whenever possible, procedures should be done using sterile techniques in a tissue culture–designated biosafety cabinet.

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

• 1Sterilize all dissection tools by autoclaving in self‐sealing sterilization bags.
• 2
  Prepare working stocks of PDL and treat plates as follows:

  • a. Dissolve PDL in 1× borate buffer to a concentration of 100 µg/ml (10× concentrated). Store ∼1‐ml aliquots at –20°C.
  • b. Dilute 10× concentrated PDL in dPBS to 10 µg/ml (1×) and filter‐sterilize.
  • c. Per brain to be cultured, treat two wells of a non‐tissue culture–treated 12‐well plate with 1.0 ml of 1× PDL per well at room temperature for ≥1 hr. Alternatively, treat plate overnight by incubating with PDL at room temperature or in a humidified 37°C incubator for use the next day.
    Ensure the volume of PDL is sufficient to fully cover the bottom of the culture area. Do not allow the plate to dry out.
  • d. Just prior to use, remove PDL from culture plate, rinse three times with sterile water, and keep in sterile water until ready to plate cells. Remove all traces of liquid by thorough aspiration prior to plating cells (see step 25).
• 3Prepare stock microglia growth medium, microglia growth medium + GM‐CSF, isotonic Percoll, and 37% isotonic Percoll if not already prepared. Filter‐sterilize and store at 4°C until use. Prepare collagenase digestion buffer immediately before tissue extraction and keep on ice or at 4°C.
• 4For each brain to be cultured, label three 15‐ml conical tubes and one 50‐ml conical tube. For each brain, fill one 15‐ml conical tube with 5 ml microglia growth medium (without GM‐CSF) for brain collection (step 12) and one with 10 ml collagenase digestion buffer for tissue digestion (step 14). Leave one 15‐ml conical tube and the 50‐ml conical tube empty for use in Percoll isolation (step 22) and for mechanical digestion of brain tissue (step 16), respectively.

• 5Place C57BL/6J mouse in an isoflurane induction chamber to deeply anesthetize.

  Before moving forward, animals should be non‐responsive to stimuli, such as toe pinch.

• 6Once animal is anesthetized, remove animal from induction chamber and place under nose cone to maintain isoflurane induction.
• 7While the animal is anesthetized, pin down its feet and spray abdominal skin and fur with 70% EtOH. Then, pinch skin with tissue forceps and make small incision with surgical scissors. Cut along the skin to expose the peritoneal cavity. Holding the xyphoid process with forceps, clip the peritoneum along the ribcage to expose the abdominal cavity. Next, using scissors, cut along the bottom of the ribcage to sever the diaphragm.
• 8Cut up the ribcage on either side to expose the lungs and heart. Fill a 20‐ml Luer‐Lok syringe with 20 ml sterile cold dPBS and cap with a 20‐G hypodermic needle. Insert the needle into the apex of the left ventricle, angling the bevel toward the right ventricle. Clip the left atrium and steadily perfuse with 20 ml sterile cold dPBS.
• 9Decapitate the animal, cut the skin up to the nose, and expose the skull. Using scissors, make a small incision in the skull between the eyes. Next, insert the bottom prong of the scissors into the base of the skull (where it meets the spinal cord) and, keeping the upper prong above the skull, make small incisions to slice up the center of the skull from the spinal cord until the scissors meet the initial incision between the eyes.
• 10Remove the skull and expose the brain. Using forceps or a dissection scoop, gently remove the brain from the base of the skull, with or without the olfactory bulb and cerebellum. Sever the dislodged brain at the spinal cord and remove brain from skull.
• 11Remove the olfactory bulbs and cerebellum and discard. Using a razor blade, slice the brain in half along the longitudinal fissure, severing the cortices. Using forceps, remove the midbrain (white tissue) from each cortex and then gently flip out the hippocampus and discard.
• 12Place both cortices into the appropriately labeled 15‐ml conical tube with 5 ml microglia growth medium (see step 4) on ice until all brains have been collected.

• 13Once all brains have been collected, decant murine cortices from collection tube into a small tissue culture dish. Using forceps, transfer cortices out of collection medium (microglia growth medium) and into the cap of a second 15‐ml conical tube.
• 14Using scissors, cut both cortices into small pieces in the cap of the conical tube and then place the tube cap on top of the labeled 15‐ml conical tube containing 10 ml collagenase digestion buffer (see step 4). Shake to suspend cortical tissue in digestion buffer.

  Cortical tissue may be cut in a tissue culture dish and then transferred into the 15‐ml conical tube if preferred; however, using the tube cap limits tissue and cell loss during transfer.

• 15Secure tube caps and place samples in a zip‐top bag with a paper towel. Gently mix at room temperature for 1 hr on orbital shaker or rocker.
• 16After 1 hr of collagenase incubation, mechanically digest cortices by placing a 70‐µm cell strainer on top of the appropriately labeled empty 50‐ml conical tube from step 4.
• 17Decant cortices along with 5 ml collagenase digestion buffer over the 70‐µm strainer and collect the collagenase digestion buffer in the 50‐ml conical tube.
• 18Using the flat end of a 5‐ml syringe plunger, gently press the tissue through the 70‐µm strainer.
• 19Rinse strainer once with the reserved 5 ml of collagenase digestion buffer.
• 20Centrifuge the digested cortices for 10 min at 500 × g, 4°C.
• 21Decant the supernatant and resuspend the cell pellet in 8 ml of 37% isotonic Percoll (see step 3).
• 22Transfer sample into final empty labeled 15‐ml conical tube from step 4.
• 23Centrifuge the sample for 30 min at 1200 × g, 4°C. Let the centrifuge stop without brake so as not to disturb the Percoll gradient.
• 24Aspirate the uppermost myelin debris layer and discard. Aspirate the 37% Percoll from the tube, leaving ∼200 µl at the bottom.

  The cells are in the pellet at the bottom.

• 25Resuspend pellet in 2 ml warm microglia growth medium + GM‐CSF (see step 3). Plate 1.0 ml cells per well of the PDL‐coated 12‐well plate from step 1.

  Typically, the cortex from one adult or aged mouse brain, i.e., two cortical hemispheres, yields ∼1 × 10⁶ viable cells. This number is sufficient for plating two wells of a 12‐well plate (∼5 × 10⁵ cells/well). Plating microglia at higher or lower densities will inhibit successful growth.

• 26Place plated cells in an incubator set at 37°C with 5% CO₂ induction for growth.
• 27Change the growth medium the day after beginning culture by removing the full volume of medium from each well, i.e., 1.0 ml, and adding an equal volume of fresh warm microglia growth medium + GM‐CSF to the side of the well to avoid disrupting the cultured cells. Perform full medium changes (with microglia growth medium + GM‐CSF) daily for the first 3 days.

  Keep GM‐CSF in the growth medium for the duration of growth, as aged microglia will die without it. Adult microglia can survive without GM‐CSF after 1 week of growth, but representative data shown in this article included GM‐CSF in the growth medium for both adult and aged microglia.

  Primary microglia cultures derived from adult or aged mice often contain more cellular debris compared to cultures from other primary cell types. This is a common and expected characteristic of these cultures and typically diminishes after several medium changes.

• 28After the first 3 days, perform full medium changes twice weekly for the lifespan of the culture.

  Cultures typically reach maturity in ∼14 days and may be maintained for up to 30 days.

• 29
  Once microglia reach 80% confluency, split cultures using standard trypsinization methods to generate sub‐cultures:

  • a. Aspirate the entire volume of medium from the wells of the 12‐well plate.
  • b. Gently add 0.5 ml sterile dPBS to the side of each well to avoid disrupting the cultured cells while washing off residual culture medium.
  • c. Aspirate to remove the entire volume of dPBS and add 0.5 ml trypsin‐EDTA.
  • d. Place cells in an incubator with 37°C with 5% CO₂ induction for 5 min to allow cells to detach. Observe detachment with a brightfield microscope.
  • e. After 5 min of trypsinization, neutralize the trypsin with 0.5 ml fresh warm microglia growth medium + GM‐CSF, bringing the total volume in the well to 1.0 ml. Transfer 0.5 ml of the total volume to a new PDL‐treated well to split at a 1:2 ratio.
• 30Perform a full medium change with warm microglia growth medium + GM‐CSF the following day, after cells re‐adhere, to remove neutralized trypsin. Continue performing full medium changes twice weekly for the lifespan of the culture.

REAGENTS AND SOLUTIONS

Collagenase digestion buffer

• 100 µl collagenase D (50 mg/ml stock diluted in HBSS; Millipore Sigma, cat. no. C0130)

• 10 µl Nα‐tosyl‐l‐lysine chloromethyl ketone hydrochloride (TLCK) trypsin inhibitor (100 µg/ml stock diluted in HBSS; Millipore Sigma, cat. no. T7254)

• 100 µl DNase I (1 mg/ml stock; Millipore Sigma, cat. no. D4263)

• 100 µl HEPES, pH 7.4 (1 M stock; Gibco, cat. no. 15630‐080)

• 9.69 ml Hanks’ Balanced Salt Solution (HBSS; 1×; diluted from 10×, no calcium, no magnesium, no phenol red; Gibco, cat. no. 14185‐052)

• Prepare fresh immediately before use

Isotonic Percoll

• 450 ml Percoll (Cytiva, cat. no. 17‐0891‐01)

• 50 ml 10× dPBS

• Filter‐sterilize

• Store ≤6 months at 4°C

Isotonic Percoll, 37%

• 74 ml isotonic Percoll (see recipe)

• 126 ml microglia growth medium (without GM‐CSF; see recipe)

• Filter‐sterilize

• Store ≤6 months at 4°C

Microglia growth medium

• 445 ml DMEM/F12 (Gibco, cat. no. 11320‐033)

• 50 ml fetal bovine serum (FBS; qualified, heat inactivated; Gibco, cat. no. 16140‐071)

• 5 ml antibiotic‐antimycotic (100×; Gibco, cat. no. 15240‐062)

• Filter‐sterilize

• Store ≤1 month at 4°C

Microglia growth medium + GM‐CSF

Add 50 µl of 5 µg/ml granulocyte‐macrophage colony‐stimulating factor (GM‐CSF; Gibco, cat. no. PMC2014) to 50‐ml aliquots of microglia growth medium (see recipe) to working concentration of 5 ng/ml. Store ≤2 weeks at 4°C.

COMMENTARY

Background Information

This protocol describes the isolation and culture of microglia from adult and aged mouse brains. Cultured primary microglia represent a valuable tool that can be used for a variety of downstream analyses and that may be superior to either immortalized cell lines or in vivo microglia for certain experimental questions. Immortalized microglia cell lines, including BV2, N9, and HMC3, among others, are widely used due to their simple maintenance, easy propagation, and consistency (Timmerman et al., 2018). However, these immortalized cell lines do not capture the heterogeneity of the microglia population or many disease‐ or age‐specific conditions. As an alternative, in vivo microglia are more biologically intact than cultured microglia, but they present unique challenges. First, brain‐resident microglia are difficult to differentiate from peripheral infiltrating monocytes; culturing microglia in vitro allows for clearer differentiation from monocytes and other cellular responses (Lee et al., 2021). Second, cultured microglia allow for greater control over the environment and can be more readily manipulated pharmacologically, whereas many experimental manipulations in vivo are constrained by the blood‐brain barrier, which blocks permeation of both toxic and therapeutic agents (Wu et al., 2023). Third, in addition to limiting pharmacologic manipulations, the protected nature of the brain restricts direct visualization of responses, unlike in in vitro cultures. Microglia undergo morphologic alterations when they become activated (Streit et al., 1988), which can be easily visualized when cultured in vitro but can be more difficult to assess within the tissue directly. Although in vivo imaging methods, including two‐photon microscopy, positron emission tomography (PET), and magnetic resonance imaging (MRI), among others, have improved in recent years, these are specialized techniques of limited utility to the broad scientific community due to the need for dedicated equipment, the limited imaging depth, and the relatively low output of molecular information (Eme‐Scolan & Dando, 2020). Each of these experimental systems is ideally suited to answer distinct experimental questions that are all needed for a full understanding of microglia and their contribution to disease processes.

An important consideration when culturing any microglia in vitro is that their phenotype is highly dependent on their microenvironment. Microglia purified from the aged brain demonstrate heightened levels of activation compared to those isolated from the adult brain (Reagin et al., 2024), suggesting molecular mechanisms of aging result in microglial activation in the aged brain at baseline. Prior studies have shown that once microglia are removed from the brain, their gene expression becomes altered within 6 hr (Gosselin et al., 2014), and they become progressively more activated while in culture (Slepko & Levi, 1996). Signals from neurons, including CXCL1, CD200, and CD22, maintain microglia in a homeostatic resting state (Biber et al., 2007). Removal of microglia from this environment, in addition to stress due to the chemical and mechanical digestion of isolation methods,+  presents trauma to the cells that leads to an activated state (Procès et al., 2024). Additionally, growth factors that are necessary for in vitro culturing, including GM‐CSF, as further discussed below (see Critical Parameters), promote activation (Yang et al., 2010). Unsurprisingly, our representative data show that by 28 days in vitro (DIV), cultured microglia tend to be in a state of greater activation compared with directly ex vivo–derived microglia, based on their cellular expression of the activation markers MHC I, MHC II, and CD68. This heightened activation in culture is also consistent with the morphological changes observed in both adult and aged cultures by 28 DIV, suggesting that prolonged in vitro culture results in artificial activation of both adult and aged microglia. However, cultured aged microglia maintain a higher level of activation compared to their adult counterparts, similar to what was observed in ex vivo–derived cells. This suggests that in the described culture method, the heightened activation state of aged microglia persists in vitro, as observed in vivo.

Critical Parameters

Critical for the culture of aged microglia is the continued inclusion of GM‐CSF in the growth medium. The protocol described above notes that after 1 week in culture, microglia from adult mice can survive without GM‐CSF; however, aged microglia begin to die when GM‐CSF is removed from the medium. In cultured microglia, GM‐CSF promotes proliferation and activation, denoted by ameboid structure and rapid phagocytosis (Giulian & Ingeman, 1988). Established protocols have noted that including GM‐CSF in embryonic mixed glia cultures increased overall yield of microglia and improved their response to inflammatory stimuli (Lee et al., 2021). Although adult microglia can successfully grow in the absence of GM‐CSF after 7 DIV, our experience developing this protocol indicated that continued inclusion of GM‐CSF in the culture medium improved survival of microglia from aged mice. As described, this method yields healthy microglia that can be maintained in culture and subcultured for up to ∼30 days. Cultured microglia reach visible maturity after 9 to 12 DIV but do continue to grow and develop out to ∼21 DIV, at which point they develop a more ameboid‐like morphology. This morphology is sustained out to 28 DIV and after subcultivation, suggesting that the cells may have reached a terminal developmental state. Due to this, we did not attempt to grow or subcultivate cultured microglia beyond 30 DIV. This does not preclude the possibility that both adult and aged cells generated from this procedure can be stably grown and subcultured beyond 30 DIV; however, it is likely that prolonged cell culture could result in further phenotypic or functional cellular adaptations.

Troubleshooting

Please see Table 1 for a list of common problems, causes, and solutions.

Table 1.

Troubleshooting Guide for Culturing Primary Microglia from Adult and Aged Mice

Step	Problem	Possible cause	Solution
15	Low cellular yields	Incomplete collagenase digestion	Ensure collagenase enzyme is fresh/not expired and diluted to the proper working stock concentration
19	Low cellular yields	Incomplete rinsing of 70‐µm membrane	Gently press brain tissue through filter until no tissue is left. Ensure no large tissue pieces remain on filter after rinsing.
26	Microglia do not adhere to plates or die after culture	Used tissue culture–treated plates	Use non‐tissue culture–treated plates for primary microglia
26	Microglia do not mature/develop	Lack of GM‐CSF in medium	Include fresh/non‐expired GM‐CSF in the growth medium for at least the first week of culture
29	Phenol red indicator in growth medium turns yellow	Microbial contamination in culture	Sterilize all dissection tools and work surfaces. Filter‐sterilize all media and solutions used during culture process. Use antibiotics and/or antimycotics in culture media. Antibiotics/antimycotics can also be added to PBS used for transcardial perfusion, if necessary.

Understanding Results

Morphology

Using the procedure presented here in the Basic Protocol, cortical tissue was harvested from both 8‐wk adult and 18‐mo aged C57BL/6J mice. Growth of adult and aged microglia was observed for 28 days by brightfield microscopy (Fig. 1). Healthy outgrowth of distinct microglia cells was observed by ∼9 DIV, and cells reached maturity at ∼12 DIV. Aged microglia appeared slower to mature than their adult counterparts, with more undifferentiated microglia observed in culture on day 9 compared to their adult counterparts. Both adult and aged microglia appeared to reach steady growth and maturity by 12 DIV. From 9 to 21 DIV, both adult and aged microglia displayed a more ramified morphology, with long branching protrusions visible by brightfield microscopy. Beyond day 21, both adult and aged microglia began to adopt a more ameboid‐like morphology, with more retracted branches and enlarged soma, which is often reflective of heightened activation and phagocytic capacity (Streit et al., 1988).

Figure 1.

Representative brightfield images of adult and aged microglia grown in culture for up to 28 days. Microglia were collected from (A) 8‐wk‐old and (B) 18‐mo‐old mice and grown in culture, as described in the Basic Protocol. Brightfield images were taken at 20× magnification on the days indicated. Cells were split 1:2 on 14 and 23 DIV. Scale bars representative of 50 µm.

Immunohistochemistry

We examined the purity of our adult and aged microglia cultures by immunohistochemistry (Fig. 2). At 23 DIV, microglia were trypsinized and subcultured on coverslips for fluorescence microscopy. Circular coverslips were acid‐etched and PDL‐coated using standard laboratory methods prior to seeding with microglia. At 24 DIV, microglia were fixed using 4% paraformaldehyde with 1% sucrose for 15 min and immunostained for common markers of microglia [Iba1 (Iba1 antibody, FUJIFILM Wako Pure Chemical Corporation, cat. no. 019‐19741), 1:1000, and TMEM119 (TMEM119 antibody, Synaptic Systems, cat. no. 400 211), 1:1000], astrocytes [GFAP (GFAP antibody, Cell Signaling Technology, cat. no. 12389), 1:1000], or neurons [NeuN (NeuN antibody, Cell Signaling Technology, cat. no. 12943), 1:1000] followed by fluorescent‐labeled secondary antibody (1:400; Alexa Fluor 647 anti‐rabbit, Life Technologies, cat. no. A‐21245) and DAPI nuclear stain (1 µg/ml; Thermo Fisher Scientific, cat. no. D1306). Coverslips were mounted to slides using ProLong Gold Antifade Mountant (Invitrogen, cat. no. P36930) and allowed to cure for 24 hr, and then images were captured using a Keyence BZX‐800 fluorescence microscope at 40× objective magnification. Three individual images were taken for each coverslip, and images were processed using Fiji‐ImageJ software (v. x86‐32, https://imagej.net/software/fiji/) (Schindelin et al., 2009). Results show that both adult and aged microglia cultures co‐stained for Iba1 and TMEM119 (Fig. 2), confirming their microglial identity. We observed no staining for GFAP or NeuN in our cultures, suggesting that the culture protocol results in the development of pure microglial cultures with no contamination with astrocytes or neurons. Additionally, Iba1 staining confirmed similar ameboid morphology for adult and aged microglia in culture, suggesting potential heightened activation or phagocytic capacity of in vitro–cultured microglia.

Figure 2.

Representative immunostained images of adult and aged microglia. Microglia were collected from (A) 8‐wk‐old and (B) 18‐mo‐old mice and grown in culture for 24 days, as described in the Basic Protocol. Cells were fixed with 4% paraformaldehyde/1% sucrose, immunostained for microglia markers TMEM119 (light blue) and Iba1 (green), and counterstained with nuclear marker DAPI (dark blue). Merged images show positive staining for TMEM119 and Iba1 and no apparent nuclei negative for these markers. Images were captured at 40× magnification. Scale bars representative of 100 µm.

Immunophenotyping

To assess the activation status of adult and aged microglia directly following isolation compared with after culturing for 21 days, we performed flow cytometry analysis. For direct ex vivo analysis, we isolated microglia from adult and aged brains using collagenase digestion and a Percoll gradient, as described in the Basic Protocol. Instead of being resuspended in growth medium, isolated cells were immunostained for common markers of microglia and macrophages [CD45 (CD45 antibody, Cytek Biosciences, cat. no. 70‐0451) and CD11b (CD11b antibody, Cytek Biosciences, cat. no. 60‐0112)] and a microglia‐specific marker [P2RY12 (P2RY12 antibody, Cytek Biosciences, cat. no. 60‐0112)] as well as MHC I (MHCI antibody, eBioscience, cat. no. 61‐5958‐82), MHC II (MHCII antibody, BioLegend, cat. no. 107602), and CD68 (CD68 antibody, BioLegend, cat. no. 137002) for assessment of activation using established methods (Fig. 3A to 3C) (Reagin et al., 2024). Alternatively, adult and aged microglia were grown according to our culture procedures for 21 days, at which point cells were trypsinized and stained for flow cytometry concurrently with microglia purified directly from the brains of adult or aged animals (Fig. 3D to 3F). Results confirmed that in the absence of in vitro culturing, aged ex vivo–derived microglia possessed higher levels of activation than their adult counterparts, as determined by expression of MHC I, MHC II, and CD68 (Fig. 3G to 3I). Flow cytometry analysis further confirmed the purity of our culturing procedure, demonstrating that the majority of the cells in both adult and aged cultures co‐expressed both CD11b and CD45, along with high levels of the microglial marker P2RY12 (Fig. 3D). Among in vitro–cultured microglia, aged cells maintained higher levels of MHC I, MHC II, and CD68 compared to adult controls (Fig. 3G to 3I); however, overall activation levels of both adult and aged microglia were higher in in vitro–cultured microglia compared to ex vivo–derived adult and aged microglia.

Figure 3.

Representative flow cytometry analysis of ex vivo derived or in vitro cultured microglia. Microglia were (A‐C) derived from direct ex vivo isolation or (D‐F) cultured in vitro, as described in the Basic Protocol, for 28 days. Flow cytometry data were collected on a BD Fortessa X‐20 equipped with BD FACSDiva software (v. 8, https://www.bdbiosciences.com/en‐us/products/software/instrument‐software/bd‐facsdiva‐software) and subsequently analyzed using FlowJo (v. 10.9.0, https://www.flowjo.com/). Microglia were identified through sequential gating of total events to exclude cellular debris and doublets, followed by verification of CD11b, CD45, and P2RY12 immunopositivity. (A and D) Representative flow cytometry plots of monocyte marker CD45^hiCD11b⁺ and microglia‐specific marker P2RY12. Representative histograms of microglia activation markers MHC I, MHC II, and CD68 from (B and C) ex vivo–purified or (E and F) in vitro–cultured microglia. Quantification for microglia activation markers was done by drawing a positive‐expression gate at the point where the positive and negative peaks diverged. From this, we quantified the percentage of microglia positivity and mean fluorescence intensity (MFI) for (G) MHC I, (H) MHC II, and (I) CD68. Two‐way ANOVA and graphical representation were done using GraphPad Prism software (v. 10.0, https://www.graphpad.com/). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Time Considerations

Preparation prior to starting the culture dissection requires 1 to 2 hr. Treating culture plates with PDL requires ≥1 hr but can alternatively be performed overnight. All buffers and media can be prepared 1 day in advance, except for the collagenase digestion buffer, which should be prepared fresh the day of use.

Dissection procedures can vary widely depending on a number of factors, including the number of mice and the proficiency of the researcher. As a general rule, each mouse will take approximately 10 to 15 min to euthanize, perfuse, and dissect, plus additional set‐up and clean‐up time.

The dissociation and culturing process requires ∼5 hr, depending on the number of samples to process and the familiarity of the researcher with the process. Once microglia are in culture, processes for splitting and sub‐culturing primary microglia are similar to those for other mammalian cell culture. The process of splitting cells or changing medium can usually be completed in <1 hr approximately twice each week. These cells remain viable in culture for up to 1 month.

Author Contributions

Katie L. Reagin: Formal analysis; investigation; methodology; visualization; writing—original draft; writing—review and editing. Rae‐Ling Lee: Investigation; methodology; visualization; writing—original draft; writing—review and editing. Kristen E. Funk: Conceptualization; funding acquisition; methodology; resources; writing—original draft; writing—review and editing.

Conflict of Interest

The authors declare no conflict of interest.

Reagin, K. L. , Lee, R. , & Funk, K. E. (2025). Novel in vitro culture of microglia from aged mice: Implications for the future of aging neurobiology research. Current Protocols, 5, e70199. doi: 10.1002/cpz1.70199

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.