Flow cytometry analysis of LAMP1 (Figure S5C)

Detection: LAMP1 fluorescence intensity measured by flow cytometry.

Cell types:
SH-SY5Y wild-type, SLC45A1 knockout (SLC45A1-KO), and rescued cells (SLC45A1-KO + SLC45A1 cDNA) cultured under serum-replete or serum-depleted conditions for 72 h.

Flow cytometry for LAMP1 staining 
Cells were lifted using trypsin and then centrifuged. The cell pellet was resuspended in 1 ml of 3% formaldehyde in PBS and incubated for 10 min at 37¡C. The cells were then centrifuged and resuspended in 90% ice-cold methanol while vortexing at low speed to prevent clump formation. The cells were incubated on ice for 30 min, briefly vortexing every 10 min. After centrifugation (~1000 g, 3 min) and decanting the supernatant, the cells were washed with PBS. Following another centrifugation, the cells were resuspended in 1-2 ml of 3% BSA in PBS and incubated for 30 min at 37¡C. After centrifugation, the cells were resuspended in 3% BSA in PBS containing a 1:1000 dilution of LAMP1 antibody (Rabbit-anti human LAMP1, Cell Signaling Technology,). The mixture was incubated for 1-2 h at 37¡C, with intermittent vortexing. After centrifugation, the cells were resuspended in the wash buffer (PBS + 1% BSA + 0.05% Tween-20) and centrifuged again. The cells were then resuspended in 3% BSA in PBS containing a 1:1000 dilution of Alexa Fluor-647 and incubated for 30 min to 1 h at 37¡C. The cells were washed twice with wash buffer, centrifuged, and finally resuspended in wash buffer. The intensity of LAMP1 was measured using flow cytometry.
