To establish an orthotopic breast tumor model, we first purified RFP+ cells from passages 10–15 of Tri-PyMT cell culture by FACS. The purified RFP+ Tri-PyMT cells (1×106 cells with purity >99%, Extended Data Fig. 5a ) were injected into the mammary fat pad of 8-week old female CB-17 SCID mice. The growth of the primary tumor was monitored by external caliper measurement once a week. In approximately 4 weeks, the primary tumor was surgically removed and the incision was closed with wound clips. The tumor size did not exceed 5% of total body weight as permitted in the IACUC protocol. Animals were sacrificed 4 weeks after primary tumor removal to analyze the development of pulmonary metastasis. For animals subjected to chemotherapy, Cyclophosphamide (CTX, Sigma-Aldrich, 100mg/kg) was administered once per week, for 2 weeks prior and 2 weeks after surgery.
Cyclophosphamide ctx
Manufactured by Merck Group
Sourced in United States
Cyclophosphamide (CTX) is a chemical compound used in various laboratory applications. It is a white, crystalline powder that is soluble in water and organic solvents. Cyclophosphamide is commonly used as a reference standard or reagent in analytical and research procedures.
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Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
2 4 dinitrofluorobenzene dnfb, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mopc 1, by BioXCell (1 mentions)
Rat igg2b, by BioXCell (1 mentions)
Mouse igg1, by BioXCell (1 mentions)
Ltf 2, by BioXCell (1 mentions)
Rb6 8c5, by BioXCell (1 mentions)
Tnf α, by Nanjing Jiancheng (1 mentions)
Anti cd16 cd32, by BD (1 mentions)
Crl 2113, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Hepes, by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 yl 5 3 carboxymethoxyphenyl 2 4 sulfophenyl 2h tetrazolium mts, by Promega (1 mentions)
Cisplatin cddp, by Merck Group (1 mentions)
Anti foxp3 pe, by BD (1 mentions)
17 protocols using cyclophosphamide ctx
1
Orthotopic Breast Tumor Model in SCID Mice
2
Antibody characterization and in vivo use
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The 314.8 mAb (mouse IgG1 anti-human ICOS) has been described before.21 Isotype controls (mouse IgG1, MOPC-1; rat IgG2b, LTF-2) and anti-Gr1 mAb (rat IgG2b, RB6-8C5) were purchased from BioXcell (West Lebanon, NH, USA). The MT807R1 recombinant Ig consisting of rhesus IgG1k constant regions and CDRs derived from the anti-human CD8 antibody M-T807 grafted into rhesus variable framework regions and was provided by the Nonhuman Primate Reagent Resource (NIH contract HHSN272200900037C and grant RR016001). The antibody was expressed in vitro using serum-free medium and purified by protein-A affinity chromatography. Endotoxin was <1EU/mg. Cyclophosphamide (CTX, Sigma Aldrich) was prepared extemporaneously according to supplier technical data sheet, i.e to 20 mg/ml of injectable water. All reagents were injected intraperitoneally.
3
Cyclophosphamide Cytokine and Biomarker Assay
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Cyclophosphamide (CTX), used as the positive drug, was purchased from Sigma (St. Lousis, MO, USA. Detecting kits for IL-2, TNF-α, VEGF, AST, ALT, CRE and BUN were purchased from Nanjing Jiancheng Biotechnology Co. (Nanjing, China).
4
Preparing Immunotherapy Reagents
5
Cyclophosphamide-Induced Rat Model
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Male Sprague-Dawley (SD) rats, age 6–8 weeks, and weighing 250–300 g (Laboratory Animal Center of Guangxi Medical University, Guangxi, China) were acclimated for 5–7 days prior to use in an environment with controlled temperature and relative humidity, and a 12-h light-dark cycle. All animal studies and experimental protocols were approved by the Guangxi Medical University Institutional Animal Care and Use Committee. Cyclophosphamide (CTX) was purchased from Sigma (St. Louis, MO, USA).
6
Murine Cancer Cell Culture and Treatments
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The EG.7-OVA thymoma (ATCC® CRL-2113™), MCA205 fibrosarcoma cells and MCA205-Tlr3−/− obtained from mice lacking Tlr-3 (kindly provided by Zitvogel, Gustave Roussy Cancer Campus, Villejuif, France), were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS, Lonza), 2 mM L-Glutamine (Lonza), 0.1 U/mL penicillin, 0.1 mg/mL streptomycin (Lonza), 10 mM HEPES, 1.0 mM sodium pyruvate (NaPir), and 0.05 mM β-mercaptoethanol (β-ME) (all from Lonza), hereafter referred to as complete RPMI, and split every three days. Gentamicin (G-418 sulphate, Gibco, 0.4 mg/mL) was added to EG.7-OVA at every medium change. The cell lines were routinely tested for the absence of mycoplasma and passaged for no more than four times from thawing. Cyclophosphamide (CTX, Sigma–Aldrich, St. Louis, MO, USA), the in vitro active analogue of CTX mafosfamide (4-thioethane sulfonic acid salt of 4-hydroxy-cyclophosphamide, MAFO, Sigma) and cisplatin (cis-diamminedichloroplatinum (II), CDDP, Sigma) were dissolved in saline and filtered sterile before use. Type I Interferon (IFN-I) was produced at the department of Oncology and Molecular Medicine as previously described [16 (link)]. A mock preparation was used as specificity control.
7
In Vitro Cytotoxicity Assay for A375 Cells
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Human MM cell line A375 was obtained from ATCC (Manassas, U.S.A.). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from Promega (Madison, U.S.A.). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Waltham, USA). Cyclophosphamide (CTX) was purchased from Sigma (St. Louis, U.S.A.).
8
Orthotopic Breast Tumor Model in SCID Mice
9
Cytotoxic Effects of Dispersant Corexit
10
Fisetin-Mediated Immunomodulation Assay
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Fisetin (>98% pure, catalog number: 528483, Figure 1 ) was obtained from Sigma-Aldrich and diluted in culture media containing 0.1% DMSO, sterile filtered, and stored at 4°C prior to utilization. An equivalent DMSO concentration was used for all control animals. Cyclophosphamide (CTX) and 2,4-dinitrofluorobenzene (DNFB) were procured from Sigma (MO, USA). RPMI-1640 was from HyClone (Logan, UT). Moreover, BioLegend (CA, USA) supplied IL-6, IL-17, TGF-β, TNF-α, and IL-10 ELISA kits. PE-anti-IL-17A, FITC-anti-CD4, APC-anti-CD25, and PE-anti-Foxp3 were from BD Pharmingen.
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