Cristal violet

2D-angiogenesis assay was performed with 4 × 10⁴ EPC per well seeded on Matrigel-coated 8-well Labtek and incubated for 16 h with 3 nM RANTES, preincubated or not with heparin (10 µg/mL), or with [⁴⁴AANA⁴⁷]-RANTES or [E66A]-RANTES. Alternatively, EPC were pre-treated for 2 h either with anti-CCR1, anti-CCR5, anti-SDC-1, anti-SDC-4, anti-CD44, or with their respective isotypes (5 µg/mL), or with beta-D-xyloside (1 mM) for 72 h. After 16 h, vascular tubes were fixed with 4% PFA, stained with 0.05% Cristal violet (Sigma- aldrich) and photographed with phase contrast microscopy (OLYMPUS CK40). The average length (in µm) and area (in µm²) of 40 vascular tubes were evaluated using the Scion Imager System (Scion Image Software and National Institutes of Health, Release Beta 3b Software).

After 48 h of treatment or cotreatment with Mevalonic acid and c-Myc inhibitor, PaCa44 and PT45 cells were detached, plated at low density in 60 mm Petri dishes and grown for twelve days. Surviving colonies were fixed and stained with Cristal Violet (0.5% in methanol) (Sigma Aldrich, Burlington, MA, USA), air-dried and analyzed with Image J. Colony formation was calculated in comparison to untreated control samples, arbitrarily set to 100.

The following reagents and Antibodies were used: Esomeprazole, 3-methylcholanthrene, DTNB, Cristal violet, Sulfasalazine and LPS (Sigma-Aldrich); sCPG (Tocris Bioscience); LysoSensor Green DND-189, BCECF-AM and CFSE (Thermo Fisher Scientific);Cisplatin (Accord Healthcare); Mouse IFN-γ, IL-4 and recombinant GM-CSF (Relia Tech GmbH), rabbit anti-human xCT, mAbs anti-human Cytokeratin 14 (LL002) and Cytokeratin 18 (LDK 18) (Abcam); rabbit anti v-ATPase (TCIRG1, Proteintech); mAb anti-human thioredoxin (clone 2B1) kindly provided by Dr F. Clarke (University of Brisbane, Australia); mAbs to human Vimentin (V9) and Smooth Muscle Actin (1A4) (Thermofisher); rat anti-mouse CD206, CD86 (AbD Serotec), F4/80, CD86-FITC, F4/80-FITC (Biolegend) and CD206-FITC (Bio-Rad Laboratories).

For the cell culture experiments the doxycycline-inducible GBM cell line LN319 expressing IDH1 wildtype (IDH1^WT) and IDH1^R132H was used. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Germany) supplemented with 10% tetracycline-free fetal calf serum (FCS) (Clontech, USA) and standard antibiotics (Gibco, Germany). 1 µM doxycycline was used (Sigma Aldrich, Germany) to induce expression. Cell proliferation was assessed using the CellTiter-Blue Assay (Promega, Germany) as described previously [32 (link)] using the FLUOstarOPTIMA (BMG Labtech, Germany) fluorometer.
For colony-forming unit assays, 2500 cells/well were seeded in a 6-well format for 10 days before colonies were fixed and stained with Cristal Violet (Sigma, Germany). For agar assays, 8000 cells/well were seeded in a 6-well format and incubated for 3 weeks before cells were stained with Cristal Violet. Images were acquired with a Cool Snap™ HQ2 digital camera (Photometrics, USA) on an Axiophot 2 microscope (Carl Zeiss, Germany). Quantification was done using ImageJ software (open source). Results are means of three repeated experiments.

Cells (7500 per well) were mixed with 0.3% noble agarose in complete growth medium as described above, plated on top of a solid field layer of 0.6% noble agarose in complete growth medium, in a 6-well plate. Cells were fed twice a week with growth medium. After 4 weeks, the colonies were fixed and dyed with Cristal Violet (0.005% in 4% formaldehyde, Sigma), washed with PBS, and imaged. Colonies in the whole plate were then counted and data was analysed by Graphpad Prism.

Cells were plated in 6-wells plates (Corning) in DMEM containing 10% FBS, 1% Pen/Strep and 1% L-glutamine (Thermo Fisher). After 24 h from plating, siRNAs were transfected into the cells using Lipofectamine 2000 (Thermo Fisher), according to the manufacturer’s instructions. Forty-eight h after transfection, cells were starved using DMEM containing 1% Pen/Strep and 1% Glutamax without FBS. After 8–10 h of starvation, 25,000 cells were seeded in Boyden Chambers (Migration Support for 24 Well Plate with Transparent PET Membrane 8.0 μM pore in size, Corning). After 16 h of migration, cells were removed from the upper side of the filter with a cotton swab, and the cells on the lower side of the filter were stained using 1% Cristal Violet (Sigma-Aldrich) and fixed with 5% glutaraldehyde (Sigma-Aldrich). The lower side of the filters were photographed with a microscope, and cells were counted in double-blind with the aid of NIH Image software.