Pe rat anti mouse cd24

Neurons and glial cells of immortalized gba^−/− and gba^+/+ neuronal cultures were separated by FACS. Cells of each genotype were labeled with FITC hamster anti-rat CD29 and PE rat anti-mouse CD24 (BD Biosciences, San Jose, CA, USA). Single-stained and unstained cells were used as a control. Cells were sorted using a BD FACSAriaII cytometer (BD Biosciences). Results were analyzed with FACSDiva software version 6.1.3 (BD Biosciences).

Cells for flow cytometry analysis were harvested and resuspended in PBS with 1% FBS at a density of 1.0×10⁶ cells/ml. Hoechst 33342 (B2261, Sigma‐Aldrich), verapamil (V4629, Sigma‐Aldrich), and reserpine (R0875, Sigma‐Aldrich) were used at a final concentration of 32 μM (Hoechst 33342), 8 μM (verapamil), and 8 μM (reserpine) for side population analysis. PE rat anti-mouse CD24 (553262, BD Biosciences) and FITC rat anti-mouse CD44 (553133, BD Biosciences) were diluted to 1:100 for the staining.

We used the following antibodies: APC Rat Anti-Mouse CD24 (BD
Bioscience, 562349), PE Rat Anti-Mouse CD24 (BD Bioscience, 553262), Anti-Mouse
CD140a (PDGFRA) FITC (eBioscience,17-1407), Anti-Mouse CD140a (PDGFRA) APC
(eBioscience,17-1401), all at a dilution of 1:1000. Cells growing in 6-well plates
were washed once with PBS and then incubated in a volume of 500 µl of basal
(N2B27) medium with antibodies for 30 min at 37 °C, in the dark. Subsequently,
cells were washed once with PBS, 300 µl Accutase (Life Technologies) was added and
cells were gently dissociated by pipetting up and down. After adding 600 µl of
basal medium the cell suspension was loaded on a flow cytometer (LSR II, BD
Bioscience) or cell sorter (FACSAria III, BD Bioscience). Cells growing in 10 cm
dishes were first dissociated and incubated in 1 ml medium with the same
incubation conditions and antibody concentrations as for adherent cells. After
staining in solution, cells were spun down, the supernatant was removed and cells
were resuspended in 1 ml of basal medium before flow cytometry or sorting.
Sorting gates for positive and negative populations were set by
comparison to the signal measured in undifferentiated mESCs. For the experiments
shown in Fig. 4b, c cells were sorted
according to quartiles of CD24 signal at 48 h or terciles of CD24 signal at
72 h.