Journal: eNeuro

Article Title: Minimizing the Ex Vivo Confounds of Cell-Isolation Techniques on Transcriptomic and Translatomic Profiles of Purified Microglia

doi: 10.1523/ENEURO.0348-21.2022

Figure Lengend Snippet: Comparison of purity and yield among microglial cell isolation techniques. A , Schematic of the experimental design. Cx3cr1-NuTRAP brains were enzymatically and mechanically dissociated to create a single-cell suspension. Different microglial sorting techniques were compared with Cell-Input for purity, yield, and transcriptomic signatures. B , Representative flow cytometry plots of immunostained single-cell suspensions from input and after each of the sorting strategies shows a distinct population of eGFP + cells (identified as Cx3cr1 + microglia) and CD11b + CD45 + cells (identified as microglia per traditional cell surface markers, as shown by IHC in Extended Data ). Assessment of the loss of eGFP in circulating CD11b + CD45 + cells 60 d after Tam induction (Extended Data ) and the relative levels of CD45 mid and CD45 high cell populations from the CD11b + CD45 + each sort method and cell input from brain (Extended Data ) were also performed to further characterize the sort fractions. Representative plots of the gating strategies used for the Tyto sort (Extended Data ), FACSAria sort (Extended Data ), and flow cytometry (Extended Data ) are given as supplements. C , D , All sort positive fractions were enriched for ( C ) eGFP + singlets and ( D ) CD11b + CD45 + singlets (as compared with input; two-way ANOVA, main effect of TRAP fraction, * p < 0.05, *** p < 0.001; Extended Data ). When comparing positive fractions, the autoMACS positive fraction had lower % eGFP + singlets as compared with all other sort methods. FACSAria had higher percentage of eGFP + singlets than all other sort methods. FACSAria had higher percentage of CD11b + CD45 + singlets as compared with all other sort methods positive fractions (two-way ANOVA, Tukey’s post hoc test, * p < 0.05). E , Microglial yield was significantly higher in the MACSQuant Tyto positive fraction as compared with the autoMACS to MACSQuant Tyto and FACSAria positive fractions (one-way ANOVA, Tukey’s post hoc test, # p < 0.01). Created with BioRender.com.

Article Snippet: Cells from a single hemisphere of a Cx3cr1-NuTRAP mouse brain were pelleted at 300 × g for 10 min at 4°C and resuspended in 90 μl of 0.5% BSA in D-PBS with 10 μl of CD11b (Microglia) MicroBeads (#130-093-636, Miltenyi Biotec) per 10 7 total cells.

Techniques: Comparison, Cell Isolation, Suspension, Flow Cytometry