35 μm nylon mesh

Flow cytometric measurements were made on freshly isolated MC or MDM obtained by differentiation for 7 days in the presence of M-CSF. Prior to flow cytometry, adherent MDM were washed twice with Hank’s balanced salt solution (HBSS) and released from the culture flask by treating with Accutase (Cell Technologies) for 30 min at 37°C. MC and MDM were resuspended at a concentration of 10⁶ cells/mL in 1 × HBSS (pH 7.2), 5 mM EDTA and 0.5% BSA and passed through a 35 μm nylon mesh (BD Biosciences) before injection on a FACSCalibur flow cytometer (Becton Dickinson). For each cell type, 20,000 events were recorded for analysis. Dot plots were created using forward scatter (FSC-H) and side scatter (SSC-H) measurements to determine the size and granularity differences. All data were analyzed using Cell Quest software.

Forty-eight hours after transfection, hNSCs expressing each of the above shRNA constructs were dissociated by Accutase (Life Science Technology), gently resuspended in KnockOut DMEM/F-12 medium, and filtered through a 35-μm nylon mesh (BD Biosciences). All samples were kept on ice before sorting. Cells were sorted at a rate of ∼3,000 events per second on a fluorescence-activated cell sorter FACSAria II (BD Biosciences). Digital data were collected using FACS Diva software (BD Biosciences). Before sorting, the nozzle, sheath and sample lines were sterilized with 70% ethanol and Diethylpyrocarbonate (DEPC)-treated water. Between running two samples, the system was cleaned with DEPC-treated water. Both 80 μm and 100 μm nozzles (BD Biosciences) were used for hNSC cell sorting. Forward-angle and side-angle light scatter were used to set the gate for live cells. Green fluorescent protein (GFP) fluorescence intensity was detected using a blue laser operating at 488 nm and a 530/30 nm band-pass filter for fluorescein isothiocyanate/GFP. hNSCs not expressing GFP were used to determine the threshold parameters for selecting cell populations with GFP signals. Sorted cells were collected in KnockOut DMEM/F-12 medium, spun down, resuspended in 700 μl QIAzol (Qiagen) and stored at −80 °C for downstream analysis.

The written consents were obtained from patients before experiment. The procedures of human tissue harvesting and handling were approved by the Ethics Committee of Eye, Ear, Nose & Throat Hospital, Fudan University (Permit Number: 2019081). Human olfactory mucosae were dissected from patients with olfactory neuroblastoma. When tumors were resected, connected olfactory mucosae were isolated from tumor tissues and kept in iced PBS. Blood cells were removed by incubation with Red Blood Cell Lysis Solution (Miltenyi, #130-094-183). Tissues were minced into small pieces and digested with 0.25% Trypsin-EDTA and 50 μg/mL DNase I. Cell pellets were collected in growth medium that was used in murine OE colony culture without addition of Matrigel and then filtered with 35-μm nylon mesh (BD Falcon). Cell suspension was resuspended in growth medium supplemented with 100 μg/mL Primocin^TM (InvivoGen, #ant-pm-1), 2 μM 616452, 500 nM A83-01 and/or 10 μM SB431542. Approximately 10000 single cells were seeded per well in low-attached 24-well plate. Colonies were visible on Day 7 and then were passaged every 10 days or based on colony growth status.