Dxflex ow cytometer

Cells were dissociated into single cells by Accutase (Thermo Fisher Scienti c) and then xed using 4% paraformaldehyde for 15 min at room temperature. Blocking and permeabilizing procedures referred to Zhao et al. protocol [16] . After the above processes, cells were suspended with 100 μL PBS containing 0.1% BSA and stained with PLZF monoclonal antibody-PE (1:200, Invitrogen, Mags.21F7) for 40 min under room temperature. Cells were then washed and resuspended with PBS containing 0.1% BSA, and the percentage of PLZF + cells was detected by a DxFLEX ow cytometer (Beckman Coulter). For the detection of haploid cells, single cells were suspended with 300 μL cold PBS containing 10% FBS and then added with 700 μL cold ethanol. After stored at -20℃ overnight, cells were resuspended with PBS containing 100 μg/mL PI (Sigma Aldrich), 100 ng/mL RNase (CWBio, China) and 0.1% Triton X-100 for 20 min at room temperature. Cells were then washed and resuspended with PBS, and the percentage of haploid cells were detected by a DxFLEX ow cytometer. For the detection of apoptotic cells, single cells were stained with PI and Annexin V using a Annexin V-EGFP Apoptosis Detection Kit (KeyGEN BioTECH, China), and the percentage of apoptotic cells were detected by a DxFLEX ow cytometer.

SH-SY5Y cells were seeded in 6-well plates at 10 6 cells/well for 24 hours and then treated with increasing MPP + concentrations for 24 hours. Cells were the gently washed with PBS and chemically detached with 0.05% trypsin/EDTA. After detachment, trypsin was quenched with complete media, cells pelleted by centrifugation and caspase activity detected with FAM-FLICA capase-1 assay kit (ImmunoChemistry Technologies, LLC, Bloomington, MN, USA) according to manufacturer's recommendations. In a nutshell, cells were stained were incubated with FLICA (1:30) for 1 hour at 37°C protected from light. Cells were then by washed with 5 volumes of 1X apoptosis wash buffer, pelleted by centrifugation, resuspended in 1X apoptosis wash buffer with 5µL of PI and incubated for 10 minutes 37°C protected from light. Data was acquired with Beckman DxFlex ow cytometer (Beckman, USA) and analyzed with CytExpert (Beckman Coulter Inc, CA, USA). Single stain controls were used to calculate compensation. PI positive cells were detected according to the operation procedure of PI staining kit (KeyGEN Biotech, NanJing,