Facscalibur cytofluorimeter

HL60 cells (10⁵) were plated in 24-well tissue culture plates (Corning Life Sciences). Cells were treated with curcumin (25 μM), C-150 (300 and 600 nM), bortezomib (20 nM), and their combinations in triplicates. After 48 hours, cells were collected and resuspended in annexin V binding buffer (0.01 M HEPES, 0.14 M NaCl, and 2.5 mM CaCl₂). Annexin V-Alexa 488 (Life Technologies, 2.5 : 100) and propidium iodide (PI) (Sigma-Aldrich, 10 μg/mL) were added to the cells and placed for 15 min in dark at room temperature. After washing, the cells were analyzed on the FACSCalibur cytofluorimeter (Becton Dickinson). Percentage of FL1 (Annexin V-Alexa 488) positive and FL3 (propidium iodide) negative early apoptotic cells and FL1 (Annexin V-Alexa 488) positive and FL3 (propidium iodide) positive late apoptotic cells was determined. For statistical significance, two-tailed Student's t-test was used.

SH-SY5Y CTR, LMNA-KD cells and TICs were analyzed by indirect immunofluorescence. The cells were harvested, washed in cold 1X PBS and fixed (1 × 10⁶ cells/ml) in a solution containing cold acetone/methanol (1:4 v/v) in 50% 1X PBS. For each sample, 1 × 10⁶ cells were incubated with the human monoclonal antibody anti-N-Myc (Santa Cruz) in medium containing 10% FBS and 0.5% Tween 20 for 1 h at room temperature. After washing in PBS, the cells were incubated with FITC-conjugated goat anti-mouse IgG (BD, Pharmingen) in PBS for 50 min. After an additional wash in PBS, the samples were measured using a FACSCalibur cytofluorimeter (Becton Dickinson). Samples incubated with IgG isotype control antibody were used as negative controls, and NB LAN-5 cells were used as a positive control. The analysis was performed using the CellQuest software package. To determine the N-Myc immunofluorescence positivity we used the method of 2% of background.

A single cell suspension was obtained through the homogenization of dissected tumors. The cell cycle analysis was performed by flow cytometry (FCM) after propidium iodide (PI) staining. To perform PI staining, the cells were stained with a solution containing 50 μg/mL PI (Sigma-Aldrich, Milan, Italy) and 75 KU/Ml RNase (Sigma-Aldrich, Milan, Italy). For both experiments, 20,000 events/samples were acquired using a FACScalibur cytofluorimeter (Becton-Dickinson, Sunnyvale, CA, USA).
Cell death was evaluated by the Trypan Blue dye exclusion test.

For the cell cycle phase distribution analysis, both adherent and floating cells were fixed in 70% EtOH and incubated at 4 °C for 30 min in staining solution containing 50 mg/mL of propidium iodide, 50 mg/mL of RNase, and 0.05% Nonidet-P40 in PBS. Samples were analyzed with a Fluorescence Activated Cell Sorting (FACS)-Calibur cytofluorimeter (Becton Dickinson, Milan, Italy). At least 30,000 events were read, and the histograms were analyzed using the CellQuest software according to the Modfit model (Becton Dickinson, Milan, Italy).

Cells were released from all cultures by digestion with 1 mg/mL collagenase IV (Sigma-Aldrich) for 30 min for 3D and 5 min for 2D at 37 °C, manually shaken in serum-free DMEM (Gibco), 12 RAFT discoids were pooled for one assay sample. Apoptosis was detected as described previously [30 (link)]. Briefly, cells cultured under different conditions were collected and resuspended in Annexin V binding buffer (0.01 M HEPES, 0.14 M NaCl and 2.5 mM CaCl₂, Sigma-Aldrich) on the 4^th and the 9^th day. Annexin V-Alexa 488 (Thermo Fisher Scientific, 2.5:100) was added to the cells, which were then kept in dark at room temperature for 15 min. Before the acquisition, propidium iodide (10 μg/mL) (Sigma-Aldrich) was added in Annexin V binding buffer to dilute Annexin V-Alexa 488 5X. Cells were analyzed on a FACSCalibur cytofluorimeter using CellQuest software (Becton Dickinson). The percentage of the FL1 (AnnexinV-Alexa 488, AnnV) negative and FL3 (propidium iodide, PI) negative living cells (AnnV−/PI−), the early apoptotic (AnnV+/PI−), late apoptotic (AnnV+/PI+) and necrotic (AnnV−/PI+) cells were determined.

For the cell cycle analysis, cells were harvested, washed with phosphate-buffered saline, and then fixed in 70 % ice-cold ethanol at 4 °C overnight. Cells were treated with RNase (final concentration 0.2 mg/ml) and stained with propidium iodide (final concentration 10 μg/ml) in the dark, following detection with a FACSCalibur Cytofluorimeter (Becton-Dickinson) as previously described (Wong et al. 2014 (link)).
For the cell apoptosis analysis, cells were harvested and stained with the Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich) following the manufacturer’s protocol. Apoptosis was measured and analyzed as previously described (Zhu et al. 2016 ). All experiments were performed at least three times.

Cells were treated with isotype control or specific antibodies for 30 min at 4 °C. Cell Signaling (Beverly, Massachusetts, USA) provided the human SOX2 antibody, while the anti-OCT4-PE antibody came from Miltenyi Biotec [43 (link)]. CellQuest software was used to evaluate cells employing a FACS Calibur cytofluorimeter (BD Biosciences, Franklin Lakes, NJ, USA; www.bdbiosciences.com). Each sample covered at least 10⁴ events that were recorded. Cells were permeabilized when required using the Cytofix/Cytoperm kit from BD Biosciences [28 (link)].
Cells were permeabilized with cold 70% ethanol in PBS for cell cycle analysis and stored at −20 °C until next day. Cells were then resuspended in PBS supplemented with 50 μg/ml propidium iodide (PI) and 100 mg/ml RNase, and were kept for 30 min at 30 °C. CellQuest software was used to analyze the data obtained through the FACS Calibur [28 (link)].
Dead cells were identified and distinguished using the Annexin V-FITC Kit from Miltenyi Biotec. Cell staining was performed in a dark environment for 15 min at room temperature by adding 10 μl of Annexin V-FITC to 10⁶ cells, following a serum starvation of 18 h. Prior to analysis employing CellQuest software and the FACS Calibur, 5 μl of PI solution was added [28 (link)].