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6 protocols using cyclophosphamide

1

Chemotherapeutic Agents Preparation

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Se-methylselenocysteine was purchased from Sigma (St Louis, MO, USA) and dissolved in sterile saline (0.9% NaCl) at a concentration of 1 mg ml−1. Cyclophosphamide and CDDP (Bristol-Myers Squibb Co., Princeton, NJ, USA) were used as a ready-to-use clinical formulation solution in 50–100 ml vials that contained 1000 mg of CTX (20 mg ml) and 100 mg of CDDP (1 mg ml−1), respectively. Oxaliplatin (ELOXATIN) was purchased from Sanofi-Synthelabo (New York, NY, USA) as a solution of 5 mg ml−1 in 10 ml vials. Irinotecan was purchased from Pfizer Inc. (New York, NY, USA) as a ready-to-use clinical formulation solution in 5 ml vials containing 100 mg of the drug (20 mg ml−1).
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2

Apoptosis Induction Protocol for Cancer Therapy

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d-luciferin, VivoGlo reagent, and goat anti-luciferase antibody were purchased from Promega. Navitoclax and venetoclax were synthesized at AbbVie as previously described (Park et al. 2008 (link); Souers et al. 2013 (link)). Cyclophosphamide was purchased from Bristol-Myers Squibb (Princeton, NJ). Doxorubicin was purchased from Bedford Laboratories (Bedford, OH). Vincristine was purchased from Mayne Pharmaceuticals (Paramus, NJ). Prednisolone was purchased from ETHEX Corp (St Louis, MO). Bendamustine was purchased from Cephalon Inc (Frazer, MA). Rituximab was purchased from Genentech (South San Francisco, CA). Phosal 50 PG was purchased from American Lecithin (Oxford, CT). Rabbit anti-cleaved caspase 3 antibody was purchased from Cell Signaling (Danvers, MA).
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3

Allogeneic Bone Marrow Transplant

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Our BMT protocol has been described [5] (link). Recipient female B10.BR mice [lethally conditioned with cyclophosphamide 120 mg/kg/d on days -3,-2 (Bristol Myers Squibb, Seattle, WA) and 7.5 Gy irradiation on day -1] were given male C57BL/6J (B6) donor BM (15×106, T-cell depleted) without or with 2×106 B6 spleen cells via caudal vein. All experiments were approved by the University of Minnesota Institutional Animal Care and Use Committee (assurance #A3456-01, IACUC # 0906A67041). The results of 5 transplant experiments were used for this study, with each experiment consisting of at least 5 mice per group.
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4

Establishing HER2+ Murine Tumor Model

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D2F2/E2 cells, a mouse mammary tumor line that has been stably transfected with a vector expressing the human HER2/neu gene and its parent cell line, D2F2 were a generous gift from Dr. Wei-Zen Wei, (Karmanos Cancer Institute, Wayne State University, Detroit, MI USA). Early passage cells were frozen and periodically thawed for experimental use or restocking. Mycoplasma testing was negative using the Impact III PCR profile from IDEXX (RADIL, Columbia, MO, USA). Anti-CTLA4 monoclonal antibody (mAb 9H10) was obtained commercially (BioXcell Fermentation/Purification Services #BE0131, West Lebanon, NH, USA) as was cyclophosphamide (Bristol-Myers Squibb Co., Princeton, NJ, USA). Mice were 8 to 20 weeks of age and weighed 20-25 g. Thy 1.2 BALB/c were obtained from Taconic (Hudson, NY). Animal studies were approved by the University of Pittsburgh institutional Animal Care and Use Committee (IACUC Protocol #: 21028761).
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5

Cyclophosphamide Intraperitoneal Injection

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Cyclophosphamide was purchased from Bristol-Myers Squibb and diluted in PBS for intraperitoneal injection (i.p.).
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6

Estradiol and Poly I:C Treatment Modulate Protein Translation and Hematopoietic Stem Cell Mobilization in Mice

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Mice were injected subcutaneously with 100 μl of corn oil containing 2 μg estradiol (Sigma, St. Louis, MO) every day for 7 days unless otherwise noted. Oil treated mice and E2 treated mice were caged separately. Poly I:C (Amersham, Piscataway, NJ) was resuspended in PBS at 50 μg/ml, and mice were injected intraperitoneally with 0.5 μg/gram of body mass every other day for 6 days. To measure protein translation, mice were injected intraperitoneally with 500 μg of puromycin (ThermoFisher Scientific, Waltham, MA) and sacrified one hour later. To promote HSC activation and mobilization, mice were intraperitoneally injected with 4 mg of cyclophosphamide (Bristol Myers Squibb, NY) on day −1, and then injected subcutaneously on successive days with 5 μg of human G-CSF (Amgen, Thousand Oaks, CA). Mice were sacrificed on day 3 or 6.
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