Non enzymatic cell dissociation buffer

DAOY cells were obtained from ATCC and cultured in MEM medium (Gibco, Invitrogen), supplemented with 10% heat-inactivated fetal bovine serum, 1% sodium pyruvate, 1% non-essential amino acid solution, 1% L-glutamine and penicillin/streptomycin. Human and mouse stem-like cells (hMB-SLC and mMB-SLC) were derived, as previously described from primary human SHH-MBs and from spontaneous tumors arisen in Ptc^+/− mice [24 (link)]. DAOY-SLCs were obtained as described in Alimova et al., 2012 [12 (link)].
All SLCs were maintained in DMEM/F12 supplemented with 0.6% glucose, 25 mg/ml insulin, 60 mg/ml N-acetyl-L-cystein, 2 mg/ml heparin, 20 ng/ml EGF, 20 ng/ml bFGF (Peprotech, Rocky Hill, NJ), 1X penicillin-streptomycin and B27 supplement without vitamin A (Gibco).
For differentiation experiments, hMB-SLCs and mMB-SLC were mechanically dissociated and plated for 48 h on D-poly-lysine coated dishes in differentiation medium (DMEM/F12 with N₂ supplement and 2 mg/ml heparin, 0,6% glucose, 60 mg/ml N-acetyl-L-cysteine, 1% FBS).
For self-renewal assessment, clonogenic assay was performed as previously described [24 (link)]. Briefly: oncospheres were disaggregated with non-enzymatic cell dissociation buffer (Sigma) and cells were plated at clonal density (1-2 cells/mm²) into 96-well plates and cultured for an appropriate number of days (7-15) until oncospheres were countable.

ATC cell lines were cultured to 70% confluence and harvested using non-enzymatic cell dissociation buffer (Sigma–Aldrich). All stainings were performed in PBS for 20–30 minutes at 4°C. Details on antibodies, clones, manufacturers, and staining conditions for FACS are listed in Supplementary Table S3. Fixable Viability Dye-eFluor506 (dilution 1:4000) was from eBioscience. For mouse thyroid tumors, stained cells were washed and fixed using the BD Cytoperm/Cytofix kit (BD Biosciences) according to the manufacturer's instructions. Samples were acquired on a BD LSR II flow cytometer (Becton Dickinson) and were analysed using FlowJo software (TreeStar). Samples from NSG mice injected with GFP^hi-expressing 8505C cells were also acquired on an ImageStreamX Mark II imaging flow cytometer and analyzed using IDEAS software (Amnis/EMD Millipore).

Cells were subcellularly fractionated using the ER isolation kit (Sigma, ER0100) as instructed by the manufacturer’s protocol. Briefly, cells plated on two 15-cm plates were detached by incubating in nonenzymatic cell dissociation buffer (Sigma). The cells were centrifuged at 300g for 3 min, resuspended in 1× hypotonic extraction buffer, and incubated at 4 °C for swelling. After 20 min, the cells were centrifuged at 600g for 5 min and resuspended in 1× isotonic extraction buffer. The cells were mechanically homogenized using a 7-ml Dounce homogenizer (10 strokes), and the lysate was centrifuged at 1000g 10 min at 4 °C for removal of nuclear fractions. The supernatants were further centrifuged at 12,000g for 15 min at 4 °C, resulting in mitochondria-enriched pellet (washed two times with PBS before analysis). For isolation of the ER, the supernatant was ultracentrifuged at 100,000g at 4 °C for 60 min, and the ER-enriched pellet was resuspended in 100 μl of isotonic extraction buffer (ER fraction), which was analyzed by immunoblot, or further incubated in the presence of freshly prepared 0.035 or 0.2% digitonin (Sigma) for 45 min at 4 °C for evaluation of the differential solubility.

BV2 cells were plated at 3×10⁵ cells per well in DMEM containing 4.5 g/L D-glucose and 10% (v/v) FBS, and incubated at 37°C overnight. DMEM was next replaced with DMEM-High or -Low (n = 6 for each group). Cells were acclimatized in the latter medium for 24 h before adding 5 mg/L of Vybrant E. coli beads (Life Technologies) in 1X Hank's balanced salts solution. Phagocytosis was allowed to proceed for 2 h at 37°C. As a negative control, phagocytosis was assessed on ice with viable cells. Cells were then washed twice with ice-cold DPBS and harvested with non-enzymatic cell dissociation buffer (Sigma-Aldrich). Non-internalized beads were quenched with 0.2% trypan blue solution (Sigma-Aldrich). BV2 cells were washed and resuspended in DPBS, and then injected into the flow cytometer. Data were acquired (at least 500 singlet events) and analyzed with the BD FACS Diva software.