Growth factor reduced phenol red free matrigel

The 8-week-old female BALB/c-nude mice (Charles River) were subcutaneously injected in the flank with 5 × 10⁵ MCF10DCIS.com cells (at P11) that had been resuspended in 400 μl of which 200 μl was growth factor reduced phenol red free matrigel (BD Bioscience) and 200 μl was vehicle (PBS) alone or in combination with human purified recombinant CLIC3 (500 ng), with or without Z-DON (20 nM). Mice were humanely euthanized after 15 days from inoculation and tumours excised and used for immunohistochemical analysis. The circularity of the tumours was determined using ImageJ. Only tumours with a peripheral laminin 5 staining were quantified. Mice were randomly assigned to a treatment group and the investigators were blinded when assessing the outcome. Mice were housed in individual ventilated cages in a barrier animal facility proactive in environmental enrichment. All animal work was done in accordance with ethical approval from University of Glasgow under the revised Animal (Scientific Procedures) Act 1986 and the EU Directive 2010/63/EU authorized through Home Office Approval (Project licence number 60/4181). Animals were excluded from the analysis if they were considered outliers based on Prism analysis.

The animal experiments were conducted according to the guidelines and ethical standards of the Animal Care and Use Ethics Committees of Sun Yat-Sen University (IACUC-DB-16-070). ADSC (100 μl, 1 × 10⁶ cells) or CRP-treated ADSC (24-hour pretreatment with 25 μg/ml CRP without FBS, 1 × 10⁶ cells) suspensions were mixed with 400 μl of ice-cold growth factor reduced phenol red-free Matrigel (BD Bioscience), and Matrigel containing PBS was used as a negative control. The Matrigel mixture was injected subcutaneously into the dorsal area of male nu/nu mice, 4–5 weeks old. Each experimental condition was performed with three mice. At day 7, the Matrigel implants were removed and then fixed with formalin, and the fixed Matrigel plug was embedded in paraffin to prepare sections for hematoxylin and eosin (H & E).