Microglia isolation from aging mice for cell culture: A beginner’s guide

Abstract

Microglia, the innate immune cell of the central nervous system, play significant roles in brain development, maintenance, homeostasis, and neuroinflammation. Although numerous methods have been developed to isolate microglia from embryonic or postnatal mouse brains, still major difficulties exist in isolating microglia from adult mice, often resulting in low yield and risk of cellular activation. Therefore, there is a need for a more efficient method to isolate pure and high-yield microglia from adult mice to study various neurodegenerative diseases. The aim of this study was to develop a fully functional protocol for the isolation of microglia by comparing different protocols. We investigated the efficacy of three protocols in terms of cell yield, purity, cellular activation, cellular aging, and migration properties and proposed the modified protocol (PROTOCOL 1), which provides an optimal yield of functional microglial cells with a minimum of material and equipment and allows young researchers with little experience to isolate microglia and helps them to delve deeper into the world of neuroscience.

Impact of microglia isolation and culture methodology on transcriptional profile and function

“…We analyzed each protocol for microglial yield, RNA yield/quality, baseline transcriptional profile, and response to HMGB1 stimulation. Our findings of greater than 90% purity across all isolation methods corroborate existing studies [ 4 , 34 , 35 ].…”

“…1 A). The adult culture protocols differ in method of isolation and time in culture prior to stimulation [ 4 , 35 ] (Fig. 1 B, C).…”

“…We adapted a previously described protocol [ 31 , 35 , 38 ], which involves a 7-day culture of adult microglia with 100 ng/mL GM-CSF and 100 ng/mL macrophage colony-stimulating factor (M-CSF). During this 7-day culture, microglia develop a ramified, adherent, reactive morphology in vitro [ 35 ].…”

“…We adapted a previously described protocol [ 31 , 35 , 38 ], which involves a 7-day culture of adult microglia with 100 ng/mL GM-CSF and 100 ng/mL macrophage colony-stimulating factor (M-CSF). During this 7-day culture, microglia develop a ramified, adherent, reactive morphology in vitro [ 35 ]. We included an additional step, whereby non-adherent contaminating cells were removed using a lower concentration trypsin and discarded to enhance purity, prior to microglial separation with higher concentration trypsin, as described in “ Methods ”.…”

“…Immediate isolation and treatment negates the effects of culture shock. However, the yield and viability of microglia obtained with this protocol is significantly less than what is obtained using the in vitro culture protocols from neonatal mice [ 35 ].…”