Nature - USA (2020-10-15)

4

nature research | reporting summary

April 2020

Ethics oversight All protocols were approved by IACUC at Icahn School of Medicine at Mount Sinai and were in accordance with NIH guidelines.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry

Plots

Confirm that:

The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).

The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).

All plots are contour plots with outliers or pseudocolor plots.

A numerical value for number of cells or percentage (with statistics) is provided.

Methodology

Sample preparation Tissue was rapidly dissected and mechanically disssociated in HBSS and centrifuged over 20% Percoll (GE Healthcare) and

mononuclear cells were collected. Cells were resuspended in FACS buffer (500mL sterile PBS, .5% BSA, 2ml 0.5M EDTA),

blocked in FC blocker (BioRad) as per manufacturer’s instructions and then stained on ice for 15 minutes with a combination

of the following antibodies: CD73-PE (12-0731-82, ThermoFisher), CD39-Alexafluor700 (24DMS1, ThermoFisher). The

samples were stained with DAPI on the final resuspension.

For FACS analysis of CD11b+ isolated neonatal microglia, forebrain was rapidly dissected and mechanically disssociated in

HBSS and centrifuged over 20% Percoll (GE Healthcare) and mononuclear cells were collected. Microglia were positively

selected for using CD11b+ magnetic beads.Cells were resuspended in FACS buffer (500mL sterile PBS, .5% BSA, 2ml 0.5M

EDTA), blocked in FC blocker (BioRad) as per manufacturer’s instructions and then stained on ice for 15 minutes with a

combination of the following antibodies: CD73-PE (12-0731-82, ThermoFisher), CD39-Alexafluor700 (24DMS1,

ThermoFisher). The samples were stained with DAPI on the final resuspension.

Instrument 2020 Attune NxT Flow Cytometer (ThermoFisher)

Software Data was collected using Attune NxT software (ThermoFisher). Data was analyzed using FCS Express Plus Software (De Novo

Software)

Cell population abundance In samples prepared from adult Cx3cr1CreErt2/+(Litt), 6% of cells were microglia based on gating on live single cells. 100% of

YFP+ cells were CD39+. 0% of cells were YFP-/CD39+. In samples prepared from adult control (CD73+/+) and CD73-/- mice,

7% of cells were microglia based on gating on live single cells and CD39+ expression. 0% of cells were CD73+ in Nt5e-/- mice.

In samples prepared from CD11b+ isolated neonatal microglia, 98.2% of cells were microglia based on gating on live single

cells and CD39+ expression.

Gating strategy Compensation was performed on single-stained samples of UltraComp eBeads (ThermoFisher), unstained beads, and

unstained cells. Forward and side scatter was used to gate on a defined population of cells to exclude debris and also select

single cells. Live cells were determined as DAPI negative. Gates to define positive and negative cells were determined using

unstained samples, fluorescence minus one (FMO) controls, in which one antibody was omitted per sample, YFP unstained

brain (YFP only), isotype control for the CD73 antibody, and CD73 /- sample.

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.