Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques

H. G. Gratzner, Alan Pollack, D. J. Ingram, R. C. Leif

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

The identification of cells in a population which is undergoing DNA replication has been accomplished in the past by autoradiographic techniques utilizing 3H thymidine. The autoradiographic methods, however, lack cytologic resolution and require relatively long periods of exposure to produce sufficient silver grains for detection. In addition, for the purposes of automated flow analysis, autoradiographic techniques are not applicable. Antibodies to 5 bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific antibodies were produced by affinity chromatography on AH Sepharose 4B to which had been coupled BrU. Anti BrU cross reacts with iododeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti BrdU treatment was followed by goat anti rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with 3H BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cells which had been pulse labeled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluorescence and peroxidase methods. Chromosomes from cells not containing BrdU did not exhibit banding.

Original languageEnglish
Pages (from-to)34-39
Number of pages6
JournalJournal of Histochemistry and Cytochemistry
Volume24
Issue number1
StatePublished - Dec 1 1976

Fingerprint

Immunologic Techniques
Bromodeoxyuridine
Chromosomes
DNA
Idoxuridine
Antibodies
Chromosome Banding
Rabbits
CHO Cells
Horseradish Peroxidase
Metaphase
Bovine Serum Albumin
Fluorescein
DNA Replication
S Phase
Affinity Chromatography
Silver
Goats
Thymidine
Peroxidase

ASJC Scopus subject areas

  • Cell Biology
  • Anatomy

Cite this

Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques. / Gratzner, H. G.; Pollack, Alan; Ingram, D. J.; Leif, R. C.

In: Journal of Histochemistry and Cytochemistry, Vol. 24, No. 1, 01.12.1976, p. 34-39.

Research output: Contribution to journalArticle

@article{8612f951b3df4fdc9410cc4af9f2d926,
title = "Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques",
abstract = "The identification of cells in a population which is undergoing DNA replication has been accomplished in the past by autoradiographic techniques utilizing 3H thymidine. The autoradiographic methods, however, lack cytologic resolution and require relatively long periods of exposure to produce sufficient silver grains for detection. In addition, for the purposes of automated flow analysis, autoradiographic techniques are not applicable. Antibodies to 5 bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific antibodies were produced by affinity chromatography on AH Sepharose 4B to which had been coupled BrU. Anti BrU cross reacts with iododeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti BrdU treatment was followed by goat anti rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with 3H BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cells which had been pulse labeled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluorescence and peroxidase methods. Chromosomes from cells not containing BrdU did not exhibit banding.",
author = "Gratzner, {H. G.} and Alan Pollack and Ingram, {D. J.} and Leif, {R. C.}",
year = "1976",
month = "12",
day = "1",
language = "English",
volume = "24",
pages = "34--39",
journal = "Journal of Histochemistry and Cytochemistry",
issn = "0022-1554",
publisher = "Histochemical Society Inc.",
number = "1",

}

TY - JOUR

T1 - Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques

AU - Gratzner, H. G.

AU - Pollack, Alan

AU - Ingram, D. J.

AU - Leif, R. C.

PY - 1976/12/1

Y1 - 1976/12/1

N2 - The identification of cells in a population which is undergoing DNA replication has been accomplished in the past by autoradiographic techniques utilizing 3H thymidine. The autoradiographic methods, however, lack cytologic resolution and require relatively long periods of exposure to produce sufficient silver grains for detection. In addition, for the purposes of automated flow analysis, autoradiographic techniques are not applicable. Antibodies to 5 bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific antibodies were produced by affinity chromatography on AH Sepharose 4B to which had been coupled BrU. Anti BrU cross reacts with iododeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti BrdU treatment was followed by goat anti rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with 3H BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cells which had been pulse labeled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluorescence and peroxidase methods. Chromosomes from cells not containing BrdU did not exhibit banding.

AB - The identification of cells in a population which is undergoing DNA replication has been accomplished in the past by autoradiographic techniques utilizing 3H thymidine. The autoradiographic methods, however, lack cytologic resolution and require relatively long periods of exposure to produce sufficient silver grains for detection. In addition, for the purposes of automated flow analysis, autoradiographic techniques are not applicable. Antibodies to 5 bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific antibodies were produced by affinity chromatography on AH Sepharose 4B to which had been coupled BrU. Anti BrU cross reacts with iododeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti BrdU treatment was followed by goat anti rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with 3H BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cells which had been pulse labeled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluorescence and peroxidase methods. Chromosomes from cells not containing BrdU did not exhibit banding.

UR - http://www.scopus.com/inward/record.url?scp=0017224871&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0017224871&partnerID=8YFLogxK

M3 - Article

C2 - 815428

AN - SCOPUS:0017224871

VL - 24

SP - 34

EP - 39

JO - Journal of Histochemistry and Cytochemistry

JF - Journal of Histochemistry and Cytochemistry

SN - 0022-1554

IS - 1

ER -