Figure 4.

The ACSA-2 epitope is expressed on all cortical astrocytes. A suspension of cortical cells from the Aldh1l1-EGFP mouse line (which is a pan-astrocyte marker line) was generated according to the standard protocol used for magnetic isolation of astrocytes. However, although we used adult brains in the experiments, we actually omitted the use of Percoll PLUS density medium, using only HBSS with calcium and magnesium. The cell suspension was initially depleted of myelin using Myelin Removal Beads II before labeling with an ACSA-2-PE antibody for flow-cytometry experiments. All EGFP-positive astrocytes were labeled with ACSA-2 antibody, indicating that the ACSA-2 epitope is present on all cortical astrocytes. Note the small population of cells labeled with ACSA-2 alone, which could be astrocytes not labeled by Aldh1l1-EGFP or other cell types that express ACSA-2 (e.g. oligodendrocytes) that are otherwise removed during our astrocyte purification procedure. This experiment was repeated four times using independent samples (effectively 1 technical replicate per sample) on separate days. At least 20,000 cells were analyzed for each sample.