An optimized protocol for the acute isolation of human microglia from autopsy brain samples

Additional Supporting Information may be found in the online version of this article.

Filename	Format	Size	Description
GLIA_21251_sm_SuppFig1.tif		1021K	Supporting Information Figure 1. White and grey matter yields different microglia purity after discontinuous Percoll separation (cell suspension B). Microglia was isolated from the subcortical white matter, cortical grey matter, capsula interna, nucleus caudatus and putamen. Microglia purity was determined by the means of flow cytometric analysis. The live cells isolated with the above described protocol from subcortical white matter and capsula interna were almost exclusively microglia, based on their CD11b/CD45 expression profile. Two populations could be observed. One with higher (labeled with an asteriks) and one with lower expression levels of CD11b/CD45 (marked with a white arrow). Next to these two populations, the protocol yielded an additional, non-microglial population (marked with a black arrow) exclusively found in samples containing grey matter (cortical grey matter, nucleus caudatus, putamen). This additional population is also obvious on the forward and side scatter scatter plots. (SSC side scatter (granularity); FSC forward scatter (size); DRAQ5 live cell marker; Specimen No. 19 case identifier, please see Table 1 for details).
GLIA_21251_sm_SuppFig2.tif		1211K	Supporting Information Figure 2. The additional population in cell suspension B from grey matter and mixed samples is sensitive to trypsinisation. Mechanical dissociation was either applied alone (A', B', C') or in combination with a trypsinisation step (A'', B'', C''). Cell suspensions at each step (A, B and C stand for cell suspension A, B and C, respectively) of the protocol were collected, stained for CD11b and CD45, and analysed by flow cytometry. Ungated contour plots are shown. Trypsinisation results in abrupt changes in the granularity and the DRAQ5 staining intensity of the additional population (the population marked by the black arrow in A' is missing in A''), suggesting that the cells membrane integrity is compromised due to the trypsin treatment. The unspecific binding of the antibodies to this population is not affected by trypsinisation (see population marked with a black arrow in A' and A'' in the ‘isotype control’ column). The size and granularity, staining profile and viability of the microglia population (labeled with an asterisk) is only marginally affected by enzymatic dissociation with trypsin (this effect can be seen in the increase of the population marked with a white arrow in C' and C''). (SSC side scatter (granularity); FSC forward scatter (size); DRAQ5 - live cell marker; Specimen No. 19 case identifier, please see Table 1 for details).
GLIA_21251_sm_SuppFig3.tif		746K	Supporting Information Figure 3. Identity of the additional population in grey matter samples. Quantitative real time PCR analysis of FACS sorted cell populations confirmed the protoplasmic astrocyte identity of the additional population in cell suspension B from grey matter samples. A Contour plots representing the microglia population (R1) and the additional population (R2) in cell suspension B. B Quantitative real time PCR analysis of the transcript levels of selected microglia (CD11b) and astrocyte (GLAST, GLT-1, Aldh1L1 and GFAP) marker genes of FACS sorted samples. (FSC forward scatter (size); SSC side scatter (granularity); for the other acronyms, please see List of Abbreviations)
GLIA_21251_sm_SuppFig4.tif		1289K	Supporting Information Figure 4. Two subpopulations of microglia can be distinguished based on their differences in apoptotic markers. The microglia subpopulation that has reduced size (R3 on FSC/SSC) shows signs of apoptotic cell death, such as increased binding to annexin V and increased staining with PI. All scatter plots show cell suspension B of a mixed (containing both white and grey matter) sample. A The microglia population was determined based on their characteristic CD11bhigh/CD45intermediate expression profile (R1). The two microglia populations were discriminated by the differences in their size (R2 and R3). R4 contains cellular debris, as shown by lack of DRAQ5 staining (G4 on the histogram). R5 comprises of the additional live cell population present only in grey matter. B The microglia of smaller size (R3) had increased binding to annexin V, a marker of early apoptotic cells, when compared to the bigger microglia population (R2) (B' no annexin V FITC added; B'' incubation with annexin V FITC). C The microglia of smaller size (R3) had increased staining with PI, a marker of late apoptotic cells, when compared to the bigger microglia population (R2) (C' no PI added; C'' incubation with PI). (FSC forward scatter (size); SSC side scatter (granularity); PE phycoerythrin; FITC fluorescein isothiocyanate; DRAQ5 a live cell marker; CD11b and CD45 microglia markers; PI propidium iodide; for the other acronyms, please see List of Abbreviations; Specimen No. 26 case identifier, please see Table 1 for details; the numbers in the corners of the scatterplots in B'/B'' and C'/C'' represent the percentage of cells in the given gate and quadrant)
GLIA_21251_sm_SuppFig5.tif		520K	Supporting Information Figure 5. The microglia subpopulation of compromised health is more pronounced in specimen from a donor of advanced age. The microglia subpopulation that has reduced size (R3 on FSC/SSC) shows signs of apoptotic cell death, such as increased binding to annexin V. All scatter plots show cell suspension B of a mixed (containing both white and grey matter) sample. A The microglia population was determined based on their characteristic CD11bhigh/CD45intermediate expression profile (R1). The two microglia populations were discriminated by the differences in their size (R2 and R3). R5 comprises of the additional live cell population present only in grey matter. B The microglia of smaller size (R3) had increased binding to annexin V, a marker of early apoptotic cells, when compared to the bigger microglia population (R2) (B' no annexin V FITC added; B'' incubation with annexin V FITC). (FSC forward scatter (size); SSC side scatter (granularity); PE phycoerythrin; FITC fluorescein isothiocyanate; DRAQ5 a live cell marker; CD11b and CD45 microglia markers; for the other acronyms, please see List of Abbreviations; Specimen No. 25 case identifier, please see Table 1 for details; the numbers in the corners of the scatterplots in B' and B'' represent the percentage of cells in the given gate and quadrant)
GLIA_21251_sm_SuppFig6.tif		855K	Supporting Information Figure 6. An immunomagnetic bead separation step after the removal of myelin/debris yields comparable purity as a discontinuous Percoll density separation step. Contour plots of flow cytometric measurements of the samples collected at the different steps of the protocol. Live cell gate was set as the DRAQ5+ cell populations (R1). The microglia population is determined as the CD11bhigh/CD45intermediate cell population (R2). In an alternative approach, approximately 80% microglia purity (determined as percentage of R2 in R1) can be attained by applying anti-CD11b antibody coupled magnetic bead separation to the cell suspension A (cell suspension A/C). (SSC side scatter (granularity); FSC forward scatter (size); DRAQ5 live cell marker; Specimen No. 17 case identifier, please see Table 2 for details).
GLIA_21251_sm_SuppTab1.doc		34K	Supporting Information Table 1. Specifications of the forward and reverse primers used for quantitative real time polymerase chain reaction analysis of the different glial transcript levels.

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