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. 2022 Mar 28;9(2):ENEURO.0348-21.2022. doi: 10.1523/ENEURO.0348-21.2022

Figure 1.

Figure 1.

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 Fig. 1-2). Assessment of the loss of eGFP in circulating CD11b+CD45+ cells 60 d after Tam induction (Extended Data Fig. 1-3) and the relative levels of CD45mid and CD45high cell populations from the CD11b+CD45+ each sort method and cell input from brain (Extended Data Fig. 1-4) were also performed to further characterize the sort fractions. Representative plots of the gating strategies used for the Tyto sort (Extended Data Fig. 1-5), FACSAria sort (Extended Data Fig. 1-6), and flow cytometry (Extended Data Fig. 1-7) 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 Fig. 1-1). 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.