BrdU

From MagnetoWiki
Jump to: navigation, search

5-Bromo-2′-deoxyuridine is a thymidine analog used as a mutagen in genetic research and as a marker of newly divided cells in neurogenesis research. Selectively incorporated into cellular DNA during S-phase.

Sigma Product Number: B5002-5G $317

Empirical Formula (Hill Notation): C9H11BrN2O5

Molecular Weight: 307.10

PubChem Substance ID:24278270

CAS Number: 59-14-3

Immunohistochemistry Protocol

Use anti-BrdU antisera (Rat Monoclonal), 0.5 mg Accurate Chem & Sci Corp #OBT0030 $729.00

Generously provided by Dr. Zuoxin Wang

BrdU Injections

50-300 μg/g body weight in 0.9% saline and 0.007N NaOH (injection volume of 0.1ml per vole).

Tissue Preparation

  1. Deeply anesthetize animal, perfuse through the ascending aorta with saline (pH 7.4) followed by cold 4% paraformaldehyde in 0.1M PBS.
  2. Postfix brain for 2-4 hrs at 4ºC, depending on the fixation.
  3. Transfer brain into 30% sucrose in PBS until it sinks.
  4. Cut brain on microtome and put sections into 0.1M PBS with 0.1% sodium azide and store at 4°C.

BrdU Immunocytochemistry

  1. Rinse 3 X 5 min in 0.1M Phosphate Buffered Saline (PBS, pH7.4).
  2. Incubate in 0.3% hydrogen peroxide and 10% methanol in 0.1M PBS for 15 min (use concentrated PBS to make solution).
  3. Rinse 5 X 5 min in 0.1M PBS.
  4. Incubate in 2N HCl in 0.1M PBS at 60°C for 30 min (use concentrated PBS to make solution).
  5. Rinse 5 X 5 min in 0.1M PBS
  6. Block in 0.5% Triton-X 100 and 10% Normal Goat Serum (NGS) in 0.1M PBS for 1 hr.
  7. Incubate in 1:4,000 dilution of monoclonal rat anti-BrdU antibody (Accurate, OBT00300X) in 0.5% Triton-X 100 and 2% NGS in 0.1M PBS at 4°C overnight.
  8. Incubate at room temperature for 1-2 hour.
  9. Rinse 5 X 5 min in 0.5% TrintonX-100 in 0.1M PBS.
  10. Incubate in 1:300 dilution of Biotin-conjugated goat anti-rat antibody in 0.5% Triton-X 100 and 2% NGS in 0.1M PBS for 2hr.
  11. Rinse 5 X 5 min in 0.1M PBS.
  12. ABC step: Vector Elite ABC Kit incubate for 90min (one drop A and one drop B in 5 ml 0.1M PBS, must be made at least 30 minutes before use to allow the formation of the avidin-biotin complex)
  13. Rinse 5 X 5 min in 0.1M PBS.
  14. Staining by DAB kit (Vector).
  15. Rinse 5 X 5 min in 0.1M PBS.


DNA Labeling with BrdUTP

Use bromodeoxyuridine triphosphate as one of the dNTPs in labeling reaction, then stain with anti-BrdU antibodies

Radiation Sensitization

Incorporation of BrdU makes cells more sensitive to radiation damage.

Toxicity

Meuth & Green PMID 4477047 "Bromodeoxyuridine triphosphate is an allosteric inhibitor of ribonucleotide reductase, and bromodeoxyuridine can therefore kill cells by starving them for deoxycytidine nucleotides."

PMID 18770731, PMID 8555370 PMID 8871927 PMID 1726743

Pharmacokinetics

Papers to look up re: pharmacokinetics

1: The toxic effect of 5-bromodeoxyuridine on cultured epithelial cells. LITTLEFIELD JW, GOULD EA. J Biol Chem. 1960 Apr;235:1129-33. No abstract available. PMID 14417553 [PubMed - OLDMEDLINE]

Related Articles Free article at journal site

2: Relative stability to thermal denaturation of deoxyribonucleic acid (DNA) preparations containing bromodeoxyuridine. KIT S, HSU TC. Biochem Biophys Res Commun. 1961 Jun 2;5:120-4. No abstract available. PMID 13756434

3: Quantitative studies of incorporation of exogenous thymidine and 5-bromodeoxyuridine into deoxyribonucleic acid of mammalian cells in vitro. KRISS JP, REVESZ L. Cancer Res. 1961 Oct;21:1141-7. No abstract available. PMID 14459708

4: Incorporation of unnatural pyrimidine bases into deoxyribonucleic acid of mammalian cells. EIDINOFF ML, CHEONG L, RICH MA. Science. 1959 Jun 5;129(3362):1550-1. PMID 13658990

5: The fate of 5-bromodeoxyuridine, 5-bromodeoxycytidine, and 5-iododeoxycytidine in man. KRISS JP, MARUYAMA Y, TUNG LA, BOND SB, REVESZ L. Cancer Res. 1963 Feb;23:260-8. No abstract available. PMID 14035826 Free article at journal site

6: Effect of 5-bromodeoxyuridine incorporation on survival of cultured mammalian cells. HAKALA MT. Biochim Biophys Acta. 1962 Nov 26;61:815-23. No abstract available. PMID 13952011 [PubMed - OLDMEDLINE]

Related Articles

7: A chromosome study of radiation sensitization by 5-bromodeoxyuridine. SOMERS CE, HUMPHREY RM. Exp Cell Res. 1963 Mar;30:208-17. No abstract available. PMID 13978156

8: Radiosensitization of a mammalian cell line with 5-bromodeoxyuridine. DELIHAS N, RICH MA, EIDINOFF ML. Radiat Res. 1962 Oct;17:479-91. No abstract available. PMID 14026711 [PubMed - OLDMEDLINE]

Related Articles

9: The distribution and fate of bromodeoxyuridine and bromodeoxycytidine in the mouse and rat. KRISS JP, REVESZ L. Cancer Res. 1962 Feb;22:254-65. No abstract available. PMID 14459709 Free article at journal site

10: Increase in radiosensitivity to ionizing radiation related to replacement of thymidine in mammalian cells with 5-bromodeoxyuridine. Dewey WC, Humphrey RM. Radiat Res. 1965 Dec;26(4):538-53. No abstract available. PMID 5849707

11: Structures of 5-bromodeoxyuridine and 5-bromouridine. Iball J, Morgan CH, Wilson HR. Nature. 1966 Mar 19;209(5029):1230-2. No abstract available. PMID 5956313

12: Intra-arterial 5-bromodeoxyuridine and x-ray therapy. Bagshaw MA, Doggett RL, Smith KC, Kaplan HS, Nelsen TS. Am J Roentgenol Radium Ther Nucl Med. 1967 Apr;99(4):886-94. No abstract available. PMID 6021392

13: Theoretical evaluation of a limitation in the use of pyrimidine analogs in radiation therapy. Suit HD. Radiology. 1966 Dec;87(6):1065-8 passim. No abstract available. PMID 5926229

14: Comparative aspects of the pharmacology of the antimetabolites. Karnofsky DA, Young CW. Fed Proc. 1967 Jul-Aug;26(4):1139-45. No abstract available. PMID 4226285

15: Loss of labelled DNA from rat brain following injections of precursors with high specific radioactivity. II. DNA labelled with 5-[131I]iodo-2'-deoxyuridine and 5-[82Br]bromo-2'-deoxyuridine. Merits I, Cain JC. Biochim Biophys Acta. 1970;209(2):327-38. No abstract available. PMID 5453521

16: The turnover of isotopically labelled DNA in vivo in developing, adult and Scrapie-affected mouse brain. Kimberlin RH, Shirt DB, Collis SC. J Neurochem. 1974 Jul;23(1):241-8. No abstract available. PMID 4859554

17: Comparison of bromodeoxyuridine labeling indices obtained from tissue sections and flow cytometry of brain tumors. Nagashima T, Hoshino T, Cho KG, Senegor M, Waldman F, Nomura K. J Neurosurg. 1988 Mar;68(3):388-92. PMID 3278075

18: Improvement of the bioavailability of the anti-herpes virus agent BVDU by use of 5'-O-alkoxycarbonyl derivatives with increased metabolic stability. Boyd MR, Cole M, Harnden MR, Luk K, Rush MA, Sutton D, Hodge RA. J Antimicrob Chemother. 1986 Oct;18 Suppl B:207-13. PMID 3793660

19: Cell kinetic studies of in situ human brain tumors with bromodeoxyuridine. Hoshino T, Nagashima T, Murovic J, Levin EM, Levin VA, Rupp SM. Cytometry. 1985 Nov;6(6):627-32. PMID 2998714

20: Bioavailability of bromodeoxyuridine in dogs and toxicity in rats. Phuphanich S, Levin VA. Cancer Res. 1985 May;45(5):2387-9. No abstract available. PMID 3986781 Free article at journal site

21: Pharmacological evaluation of intravenous delivery of 5-bromodeoxyuridine to patients with brain tumors. Russo A, Gianni L, Kinsella TJ, Klecker RW Jr, Jenkins J, Rowland J, Glatstein E, Mitchell JB, Collins J, Myers C. Cancer Res. 1984 Apr;44(4):1702-5. PMID 6704976 Free article at journal site

22: Incorporation and persistence of 5-bromodeoxyuridine in newborn rat tissue DNA. Likhachev AJ, Tomatis L, Margison GP. Chem Biol Interact. 1983 Aug 15;46(1):31-8. PMID 6616707

23: Incorporation of 5-bromodeoxyuridine into DNA in newborn rat tissues. Margison GP, Likhachev AJ, Tomatis L. Chem Biol Interact. 1980 Jun;30(3):297-303. PMID 7379209

24: Tumor uptake of radiolabeled pyrimidine bases and pyrimidine nucleosides in animal models--III. 2'-[82Br]-bromo-2'-deoxyuridine. Lee YW, Knaus EE, Wiebe LI. Int J Nucl Med Biol. 1979;6(2):109-112. No abstract available. PMID 113355

25: Bromodeoxyuridine incorporation into DNA of human leukemia cells is not concentration dependent. Powell BL, Gregory BW, Kute TE, Morgan TM, Lyerly ES, Capizzi RL. Cytometry. 1990;11(3):438-41. PMID 2340778

26: Immunocytochemical detection of 5'-bromodeoxyuridine incorporation in the central nervous system of the mouse. del Rio JA, Soriano E. Brain Res Dev Brain Res. 1989 Oct 1;49(2):311-7. PMID 2805336

27: In situ analysis of cell kinetics in human brain tumors. A comparative immunocytochemical study of S phase cells by a new in vitro bromodeoxyuridine-labeling technique, and of proliferating pool cells by monoclonal antibody Ki-67. Morimura T, Kitz K, Budka H. Acta Neuropathol. 1989;77(3):276-82. PMID 2922990

28: Steady-state arterial and hepatic venous plasma concentrations of 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine in animals--drugs which are subject to both splanchnic and extra-splanchnic elimination. Wagner JG, Stetson PL, Knol JA, Andrews JC, Walker-Andrews S, Knutsen CA, Johnson N, Prieskorn D, Terrio P, Yang Z, et al. Sel Cancer Ther. 1989 Winter;5(4):193-203. PMID 2623384

29: Tissue-specific pharmacodynamics of 5-bromo-2'-deoxyuridine incorporation into DNA in VX2 tumor-bearing rabbits. Stetson PL, Maybaum J, Wagner JG, Averill DR, Wollner IS, Knol JA, Johnson NJ, Yang ZM, Preiskorn D, Smith P, et al. Cancer Res. 1988 Dec 1;48(23):6900-5. PMID 3180098 Free article at journal site

30: Persistence of 5-bromo-2'-deoxyuridine in tissues of rats after exposure in early life. Ward JM, Henneman JR, Osipova GYu, Anisimov VN. Toxicology. 1991;70(3):345-52. PMID 1771641

31: cis-trans-isomerization of [E]-5-(2-bromovinyl)-2,2'-anhydrouridine in vivo in rats. Szinai I, Veres Z, Szabolcs A, Gács-Baitz E, Ujszászy K, Dénes G. Xenobiotica. 1991 Mar;21(3):359-69. PMID 1650515

32: The dependence of halogenated pyrimidine incorporation and radiosensitization on the duration of drug exposure. Lawrence TS, Davis MA, Maybaum J, Stetson PL, Ensminger WD. Int J Radiat Oncol Biol Phys. 1990 Jun;18(6):1393-8. PMID 2370189

33: Cyclization-activated prodrugs. Basic esters of 5-bromo-2'-deoxyuridine. Saari WS, Schwering JE, Lyle PA, Smith SJ, Engelhardt EL. J Med Chem. 1990 Sep;33(9):2590-5. PMID 2391697

34: Effect of lithium on incorporation of bromodeoxyuridine and tritiated thymidine into human parathyroid cells. Saxe A, Gibson G. Arch Surg. 1993 Aug;128(8):865-9. PMID 8343059

35: Evaluation of a new lipophilic prodrug 3', 5'-dioctanoyl-5-bromodeoxyuridine (BrdU-C8) suspended in Lipiodol as a radiosensitizer for the treatment of AH136B tumor. Ohara C, Beppu T, Yamaguchi Y, Masuda Y, Fukushima S, Kawaguchi T, Ogawa M. Anticancer Res. 1993 May-Jun;13(3):655-60. PMID 8391245

36: Measurement of incorporation of bromodeoxyuridine into DNA by high performance liquid chromatography using a novel fluorescent labelling technique. Stratford MR, Dennis MF. Int J Radiat Oncol Biol Phys. 1992;22(3):485-7. PMID 1735684

37: Kinetics of bromodeoxyuridine elimination from human colon cancer cells in vitro and in vivo. Lawrence TS, Davis MA, Stetson PL, Maybaum J, Ensminger WD. Cancer Res. 1994 Jun 1;54(11):2964-8. PMID 8187082 Free article at journal site

38: Differential expressions of the topoisomerase II alpha and II beta mRNAs in developing rat brain. Watanabe M, Tsutsui K, Tsutsui K, Inoue Y. Neurosci Res. 1994 Feb;19(1):51-7. PMID 8008235

39: Preparation, characterization and release of microencapsulated bromodeoxyuridine. Maysinger D, Filipović-Grcić J, Alebić-Kolbah T. Life Sci. 1994;54(1):27-34. PMID 8255166

40: Regional differences in bromodeoxyuridine uptake, expression of Ki-67 protein, and nucleolar organizer region counts in glioblastoma multiforme. Onda K, Davis RL, Wilson CB, Hoshino T. Acta Neuropathol. 1994;87(6):586-93. PMID 8091951

41: The application of 5-bromodeoxyuridine in the management of CNS tumors. Freese A, O'Rourke D, Judy K, O'Connor MJ. J Neurooncol. 1994;20(1):81-95. Review. PMID 7807187

42: Comparison of bromodeoxyuridine uptake and MIB 1 immunoreactivity in medulloblastomas determined with single and double immunohistochemical staining methods. Onda K, Davis RL, Edwards MS. J Neurooncol. 1996 Aug;29(2):129-36. PMID 8858517

43: Incorporation of 5-iodo-2'-deoxyuridine and 5-bromo-2'-deoxyuridine into rodent DNA as determined by neutron activation analysis. Kitchin KT, Brown JL. Anal Biochem. 1995 Aug 10;229(2):180-7. PMID 7485970

44: Pharmacokinetics and bioavailability of 5-ethyl-2'-deoxyuridine and its novel (5R,6R)-5-bromo-6-ethoxy-5,6-dihydro prodrugs in mice. Cheraghali AM, Kumar R, Knaus EE, Wiebe LI. Drug Metab Dispos. 1995 Feb;23(2):223-6. PMID 7736915

45: Long-term oral application of 5-bromo-2-deoxyuridine does not reliably label proliferating immune cells in the LEW rat. Jecker P, Beuleke A, Dressendörfer I, Pabst R, Westermann J. J Histochem Cytochem. 1997 Mar;45(3):393-401. PMID 9071321 Free article at journal site

46: Fluorescein-labeled tyramide strongly enhances the detection of low bromodeoxyuridine incorporation levels. Van Heusden J, de Jong P, Ramaekers F, Bruwiere H, Borgers M, Smets G. J Histochem Cytochem. 1997 Feb;45(2):315-9. PMID 9016320 Free article at journal site

47: Regional pharmacokinetics of 5-bromo-2'-deoxyuridine and 5-fluorouracil in dogs: hepatic arterial versus portal venous infusions. Kuan HY, Smith DE, Ensmiger WD, Knol JA, DeRemer SJ, Yang Z, Stetson PL. Cancer Res. 1996 Oct 15;56(20):4724-7. PMID 8840990 Free article at journal site

48: Bromodeoxyuridine uptake by early liver metastases in rats: a comparison of the hepatic artery and portal vein infusion routes. Ishida H, Iwama T, Yoshinaga K, Gonda T, Idezuki Y. Surg Today. 1998;28(8):822-9. PMID 9719004

49: Assessment of correlation between skin target site free drug concentration and the in vivo topical antiviral efficacy in hairless mice for (E)-5-(2-bromovinyl)-2'-deoxyuridine and acyclovir formulations. Afouna MI, Mehta SC, Ghanem AH, Higuchi WI, Kern ER, De Clercq E, El-Shattawy HH. J Pharm Sci. 1998 Aug;87(8):917-21. PMID 9687333

50: Changes in cell-cycle kinetics during the development and evolution of primate neocortex. Kornack DR, Rakic P. Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):1242-6. PMID 9448316 Free article in PMC | at journal site

51: Bromodeoxyuridine alternating with radiation for advanced uterine cervix cancer: a phase I and drug incorporation study. Eisbruch A, Robertson JM, Johnston CM, Tworek J, Reynolds KR, Roberts JA, Lawrence TS. J Clin Oncol. 1999 Jan;17(1):31-40. PMID 10458215

52: Determination of (E)-5-(2-bromovinyl)-2'-deoxyuridine in plasma and urine by capillary electrophoresis. Olgemöller J, Hempel G, Boos J, Blaschke G. J Chromatogr B Biomed Sci Appl. 1999 Apr 16;726(1-2):261-8. PMID 10348194

53: Elimination of nonspecific radioactivity from [76Br]bromide in PET study with [76Br]bromodeoxyuridine. Lu L, Bergström M, Fasth KJ, Wu F, Eriksson B, Långström B. Nucl Med Biol. 1999 Oct;26(7):795-802. PMID 10628559

54: [76Br]Bromodeoxyuridine, a potential tracer for the measurement of cell proliferation by positron emission tomography, in vitro and in vivo studies in mice. Ryser JE, Bläuenstein P, Rémy N, Weinreich R, Hasler PH, Novak-Hofer I, Schubiger PA. Nucl Med Biol. 1999 Aug;26(6):673-9. PMID 10587106

55: Analysis of 76Br-BrdU in DNA of brain tumors after a PET study does not support its use as a proliferation marker. Gudjonssona O, Bergström M, Kristjansson S, Wu F, Nyberg G, Fasth KJ, Långström B. Nucl Med Biol. 2001 Jan;28(1):59-65. PMID 11182565

56: [76Br]Bromodeoxyuridine PET in tumor-bearing animals. Gardelle O, Roelcke U, Vontobel P, Crompton NE, Guenther I, Bläuenstein P, Schubiger AP, Blattmann H, Ryser JE, Leenders KL, Kaser-Hotz B. Nucl Med Biol. 2001 Jan;28(1):51-7. PMID 11182564

57: Use of 5-[(76)Br]bromo-2'-fluoro-2'-deoxyuridine as a ligand for tumour proliferation: validation in an animal tumour model. Borbath I, Grégoire V, Bergström M, Laryea D, Långström B, Pauwels S. Eur J Nucl Med Mol Imaging. 2002 Jan;29(1):19-27. Epub 2001 Nov 22. PMID 11807603

58: A method for pulse and chase BrdU-labeling of early chick embryos. Hämmerle B, Tejedor FJ. J Neurosci Methods. 2002 Dec 31;122(1):59-64. PMID 12535764

59: Label-retaining cells in the rat pancreas: location and differentiation potential in vitro. Duvillié B, Attali M, Aiello V, Quemeneur E, Scharfmann R. Diabetes. 2003 Aug;52(8):2035-42. PMID 12882920 Free article at journal site

60: Brivudine: a herpes virostatic with rapid antiviral activity and once-daily dosing. Rabasseda X. Drugs Today (Barc). 2003 May;39(5):359-71. Review. PMID 12861349

61: Visualization of proliferating cells in the adult mammalian brain with the aid of ribonucleotide reductase. Zhu H, Wang ZY, Hansson HA. Brain Res. 2003 Jul 11;977(2):180-9. PMID 12834878

62: Tumor distribution of bromodeoxyuridine-labeled cells is strongly dose dependent. Kyle AH, Huxham LA, Baker JH, Burston HE, Minchinton AI. Cancer Res. 2003 Sep 15;63(18):5707-11. PMID 14522888 Free article at journal site

63: Direct assessment of drug penetration into tissue using a novel application of three-dimensional cell culture. Kyle AH, Huxham LA, Chiam AS, Sim DH, Minchinton AI. Cancer Res. 2004 Sep 1;64(17):6304-9. PMID 15342419 Free article at journal site

64: Thymidine analogs are transferred from prelabeled donor to host cells in the central nervous system after transplantation: a word of caution. Burns TC, Ortiz-González XR, Gutiérrez-Pérez M, Keene CD, Sharda R, Demorest ZL, Jiang Y, Nelson-Holte M, Soriano M, Nakagawa Y, Luquin MR, Garcia-Verdugo JM, Prósper F, Low WC, Verfaillie CM. Stem Cells. 2006 Apr;24(4):1121-7. Epub 2005 Dec 22. PMID 16373692 Free article at journal site

65: Gadofluorine m uptake in stem cells as a new magnetic resonance imaging tracking method: an in vitro and in vivo study. Giesel FL, Stroick M, Griebe M, Tröster H, von der Lieth CW, Requardt M, Rius M, Essig M, Kauczor HU, Hennerici MG, Fatar M. Invest Radiol. 2006 Dec;41(12):868-73. PMID 17099425

66: Ki-67 correlates with in vivo bromodeoxyuridine labeling index in operable breast cancer. Goodson WH, Moore DH, Waldman FM. J Clin Oncol. 2006 Aug 10;24(23):3809; author reply 3809. No abstract available. PMID 16896010

67: An ELISA-based method for the quantification of incorporated BrdU as a measure of cell proliferation in vivo. Behl B, Klos M, Serr M, Ebert U, Janson B, Drescher K, Gross G, Schoemaker H. J Neurosci Methods. 2006 Nov 15;158(1):37-49. Epub 2006 Jun 15. PMID 16780957

68: Dienogest inhibits BrdU uptake with G0/G1 arrest in cultured endometriotic stromal cells. Fu L, Osuga Y, Morimoto C, Hirata T, Hirota Y, Yano T, Taketani Y. Fertil Steril. 2008 May;89(5 Suppl):1344-7. Epub 2007 May 23. PMID 17511992

69: Bromodeoxyuridine labeling index as an indicator of early tumor response to preoperative radiotherapy in patients with rectal cancer. Gasinska A, Skolyszewski J, Popiela T, Richter P, Darasz Z, Nowak K, Niemiec J, Biesaga B, Adamczyk A, Bucki K, Malecki K, Reinfuss M, Kowalska T. J Gastrointest Surg. 2007 Apr;11(4):520-8. PMID 17436139 Free article in PMC | at journal site

70: The origin of deoxynucleosides in brain: implications for the study of neurogenesis and stem cell therapy. Spector R, Johanson CE. Pharm Res. 2007 May;24(5):859-67. Epub 2007 Mar 20. Review. PMID 17372683