CD171 (L1CAM) Antibody, anti-human, PE-Vio® 770, REAfinity™

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	>20 × 105 disaggregated sphere cells were stained with a stem cell‐specific antibody panel and analysed on a BDTM LSR II flow cytometer. To a volume of 100 μL cell suspension, 5 μL antibody mix was added, mixed and incubated for 20 minutes at 4°C in the dark and washed twice with 250 μL DPBS or FACS buffer.

Protocol tips
>20 × 105 disaggregated sphere cells were stained with a stem cell‐specific antibody panel and analysed on a BDTM LSR II flow cytometer. To a volume of 100 μL cell suspension, 5 μL antibody mix was added, mixed and incubated for 20 minutes at 4°C in the dark and washed twice with 250 μL DPBS or FACS buffer.

Publication protocol

"1. Cell count
The expression of stem cell markers was assessed by flow cytometry. Cells were harvested and counted in a volume of 100 μL on a BD LSR II flow cytometer (Becton Dickinson, San Diego, CA, USA) using TruCountTM tubes (BD Biosciences, San José, CA, USA). The absolute count of the cell population was obtained using FlowJo Analysis Software V10 (Tree Star, Ashland, OR, USA).

2. Instrumentation
A flow cytometer (LSR II; BD Biosciences) equipped with Diva V8.0 software was used for 9‐color phenotypic analysis. Laser alignment (405‐nm violet laser, 488‐nm blue laser, 640‐nm red laser) was verified with beads prior to running tumor cell samples. The nine colors used in the analysis were compensated twice to account for spectral overlap emitted by the fluorochromes within each laser as well as across the lasers using a semi‐automated as well as a manual process by the application of compensation beads (BD Biosciences) coated with anti‐human Ig antibodies. The beads were incubated with each individual antibody used in the nine‐color panel for 15 minutes at room temperature. Unstained and stained beads were run individually, 5000 events were recorded, and data were imported into Diva V8.0 software including compensation matrices for automatic as well as additional manual compensation.

3. 9‐color tumor cell labeling
For the evaluation of the stem cell phenotype of the gliomaspheres, >20 × 105 disaggregated sphere cells were stained with a stem cell‐specific antibody panel and analysed on a BDTM LSR II flow cytometer. To a volume of 100 μL cell suspension, 5 μL antibody mix was added, mixed and incubated for 20 minutes at 4°C in the dark and washed twice with 250 μL DPBS or FACS buffer. For the full set of antibodies see Table S1. 7‐AAD was used as viability dye. Additionally, unstained controls as well as appropriate human isotype controls were added to the panel according to the manufacturer's recommendations."

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