Anti nestin

To quantify the level of the marker nestin, SC-ASCs and HPL-ASCs were stained with the specific antibody and evaluated by flow cytometry. In particular, both cell groups were treated with eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (ThermoFisher Scientific) following manufacturer instructions. After being fixed and permeabilized, 500,000 cells were suspended in 100 µL of antibody solution consisting of the antibody anti-nestin (1:20, BioLegend, PE-labeled) diluted in MACS buffer (Miltenyi Biotec) and incubated for 10 min at 4 °C in the dark. The cell surface phenotype was subsequently assessed by CytoFLEX flow cytometer (Beckman Coulter), recording at least 100,000 events in the selected gate. For both groups, unstained cells were used as reference for autofluorescence. We recorded the parameters regarding cell dimension (FSC-A) and median fluorescence intensity (MFI) for both unstained and stained samples. The ratio of ΔMFI was calculated by dividing HPL-ASCs (stained MFI—unstained MFI) for SC-ASCs (stained MFI—unstained MFI) and used as indicator for antigen amount. Statistical analysis was performed with t Student’s test between the control group SC-ASCs and HPL-ASCs.

Early (AnnexinV positive) and Late (AnnexinV-Propidium Iodide (PI) positive) apoptotic cells were assessed with flow cytometry after 12/24/48h from treatment. UW-228, ONS-76, D425med, D283 and D341 cells were seeded at a density of 5.0x10⁴ cells/well, and treated with DSF-Cu⁺⁺ 150nM and 2uM. Cells were trypsinized and harvested, washed with PBS, incubated with AnnexinV Ab and PI (Invitrogen, Carlsbad, CA, USA), and analyzed. Cell proliferation and cycle were measured by Ki67 and PI expression. Cells were seeded at 2.5x10⁴/well, treated after 24h with 150/300nM DSF-Cu⁺⁺, and harvested after 24/48h of treatment. Pellets were washed with PBS, fixed and permeabilized with TritonX-100, and stained with anti-Ki67 antibody (Invitrogen, Carlsbad, CA, USA) and PI/RNase solution (FxCycle^™, Thermo-Fisher Scientific, Waltham, MA, USA). For cancer stem-cell (CSC) identification, anti-CD133 antibody (BioLegend, San Diego, CA, USA), anti-Nestin (BioLegend, San Diego, CA, USA), and ALDEFLUOR kit (Stem Cell Tech., Durham, NC, USA) were used. Cells were prepared and analyzed with BD FACS Celesta (BD Biosciences; Becton, Dickinson and Company, San Jose, CA, USA), and data analyzed with FlowJo software (FlowJo, LLC).

Neurospheres were transferred to 12‐well plates and washed three times with PBS. After PBS aspiration, the spheres were fixed with of 4% paraformaldehyde (PFA) for 20 minutes at room temperature and then washed three times with PBS. The spheres were blocked by PBS containing 5% normal serum and 0.025% (wt/vol) Triton X‐100 for 30 minutes and then subjected to incubation with primary and secondary antibodies at 4°C. The following antibodies were used: anti‐GFAP (Millipore, Burlington, Massachusetts; MAB360; 1:100 dilution), anti‐Nestin (BioLegend, San Diego, California; 839 801; 1:100 dilution), anti‐Mouse IgG, Alexa Fluor 488 (Thermo Fisher Scientific, Massachusetts; A21202; 1:200), and anti‐Rabbit IgG, Alexa Fluor 594 (Thermo Fisher Scientific, A21207; 1:200). Nuclear staining was performed with Hoechst 33342 (1:1 000 dilution), and the spheres were observed using a Leica DM IRB microscope.