All animal studies were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University and were conducted using female mice. All mice were fed normal diet and water ad libitum. They were maintained in the normal 12-hour light/12-hour dark cycle. All animals were closely monitored for any distress or pain throughout the study period. The weight range of animals during the study was 20 to 30 g. Strains of mice used in all studies were athymic nude (Charles River Laboratories, Frederick, MD), NSG (Sydney Kimmel Comprehensive Cancer Center colony, Johns Hopkins University School of Medicine, Baltimore, MD), and FVB/N (Jackson laboratory, Bar Harbor, ME); all mice were aged 6 to 8 weeks. The characteristics of cell lines and mice used are provided above and in tables S3 and S6. A schematic of the xenograft tumor study design is provided in Fig. 1E. An overview of the numbers of mice divided by strain and group used for the studies is provided in table S10. Depending on cohort, 3 × 106 MDA-MB-231 or HCC1954 or 5 × 106 MCF-7(HER/neo) or BT474 cells were suspended in 50 μl of PBS and Matrigel (1:1) and injected into the fourth mammary gland on either side of female mice under anesthesia. For MCF-7(HER/neo) and for BT474 xenograft studies, mice received estrogen by implanting a 60-day release estrogen pellet (0.72 mg per pellet; Innovative Research of America, Sarasota, FL) 5 days before cell line injection on the dorsal neck region through a small subcutaneous insertion made using sterile scissors while mice were under ketamine/xylazine anesthesia[ketamine (10 mg/ml) Vedco Inc., St. Joseph, MO] and xylazine (2 mg/ml; Lloyd Inc., Shenandoah, IA) mixed in sterile PBS and intraperitoneally injected at 0.01 ml/g body weight. Tumor volume was monitored by caliper measurements twice weekly when tumors became palpable. When the measured tumor volume was 125 to 200 mm3, mice were randomly assigned into cohorts containing five animals in each group. Group I received intravenous (tail vein) injections of PBS and served as (negative) control. Group II received intravenous injections to tail vein of BP (5 mg of Fe per animal), and group III received intravenous tail vein injections of BH (5 mg of Fe per animal). Group IV received injections of BNF-IgG (intravenous tail vein injections; 5 mg of Fe per animal) only for mice bearing either MDA-MB-231 or HCC1954 xenografts. The total volume of injection was 150 μl in all cases. Twenty-four hours after injection, all mice were euthanized to collect tumors and liver for analysis.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.