Abstract
Morphological and functional characterizations of cultured microglia are essential for the improved understanding of their roles in neuronal health and disease. Although some studies (phenotype analysis, phagocytosis) can be carried out in mixed or microglia-enriched cultures, in others (gene expression) pure microglia must be used. If the use of genetically modified microglial cells is not feasible, isolation of resident microglia from nervous tissue must be carried out. In this study, mixed primary cultures were established from the forebrains of newborn rats. Secondary microglia-enriched cultures were then prepared by shaking off these cells from the primary cultures, which were subsequently used to establish tertiary cultures by further shaking off the easily detachable microglia. The composition of these cultures was quantitatively analyzed by immunocytochemistry of microglia-, astrocyte-, oligodendrocyte- and neuron-specific markers to determine yield and purity. Microglia were quantitatively characterized regarding morphological and proliferation aspects. Secondary and tertiary cultures typically exhibited 73.3% ± 17.8% and 93.1% ± 6.0% purity for microglia, respectively, although the total number of microglia in the latter was much smaller. One in seven attempts of culturing the tertiary cultures had ~99% purity for microglia. The overall yield from the number of cells plated at DIV0 to the Iba1-positive microglia in tertiary cultures was ~1%. Astrocytic and neuronal contamination progressively decreased during subcloning, while oligodendrocytes were found sporadically throughout culturing. Although the tertiary microglia cultures had a low yield, they produced consistently high purity for microglia; after validation, such cultures are suitable for purity-sensitive functional screenings (gene/protein expression).
Highlights
Microglia, the resident immune cells of the central nervous system (CNS), are derived from macrophage-like cells of mesodermal origin and play important roles in both physiological and pathophysiological con ditions (Kettenmann et al, 2011; Prinz and Priller, 2014)
The amount and quality of such pro- and Abbreviations: ANOVA, One-way analysis of variance; cyclic nucleotide 3′-phosphodiesterase (CNPase), 2′,3′-Cyclic nucleotide 3′-phosphodiesterase; CNS, Central nervous system; DIV, Day(s) in vitro; Dulbecco’s Modified Ea gle’s Medium (DMEM), Dulbecco’s Modified Eagle’s Medium; fetal bovine serum (FBS), Fetal bovine serum; fluorescein isothiocyanate (FITC), Fluorescein isothiocyanate; glial fibrillary acidic protein (GFAP), Glial fibrillary acidic protein; ionized calcium-binding adapter molecule 1 (Iba1), Ionized calciumbinding adapter molecule 1; Ki67, Proliferation marker antigen identified by the monoclonal antibody Ki67; PBS, Phosphate buffered saline; proliferation index (PI), Proliferation index; PVP, Polyvinylpyrrolidone; Rpm, Revolutions per minute; room temperature (RT), Room temperature; S1, S2, Secondary subcultures; S.D., Standard deviation; subDIV, Subcloned day(s) in vitro; T1, T2, Tertiary subcultures; transformation index (TI), Transformation index
The filtered cell suspension was centrifuged for 10 min at 1000×g at RT and the pellet was resuspended in 5 ml DMEM/10% FBS, after which the primary mixed cells were seeded in the same medium (DIV0) either on poly-L-lysine-coated culture flasks (75 cm2; 107 cells/flask) or coverslips (15 ×15 mm; 2 ×105 cells/coverslip)
Summary
The resident immune cells of the central nervous system (CNS), are derived from macrophage-like cells of mesodermal origin and play important roles in both physiological and pathophysiological con ditions (Kettenmann et al, 2011; Prinz and Priller, 2014). The methods used for isolating microglia from nervous tissues or for subculturing from a mixed neuronal/glial culture include differential adhesion (shaking) techniques, gradient density centrifugation (Gingras et al, 2007; Moussaud and Draheim, 2010; Jin and Kim, 2015), cell sorting (Ford et al, 1995; Marek et al, 2008) and harvesting microglia from a non-adherent floating cell layer without any shaking at all (Moussaud and Draheim, 2010) As many of these often-controversial or not fully documented methodological studies used different separation techniques or were carried out without an extensive quantitative analysis of the contami nating cell types present in the cultures, we set out to analyze meticu lously our differential adhesion culture method, emphasizing the critical steps that are necessary for the establishment of highly enriched microglial cultures; we aimed to catalog all of the major contami nating cell types. Such detailed description of microglial cell cul tures established from neonatal rats is unique in the literature
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
