Analysis of thymic lymphocyte apoptosis using in vitro techniques

Khaled Machaca, Mark M. Compton

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Thymocyte apoptosis was analyzed in 4-week-old chicks that were treated with dexamethasone. Glucocorticoid treatment of birds resulted in the internucleosomal cleavage of thymocyte DNA as detected by agarose gel electrophoresis and the generation of a population of thymocyte nuclei with a hypodiploid content of cellular DNA when analyzed by flow cytometry. Visualization of apoptotic thymocytes at the ultrastructural level or via confocal microscopy revealed the typical morphological characteristics of cells undergoing apoptosis. Similar techniques were employed to further analyze apoptosis using an in vitro approach where extracts of thymocyte nuclear proteins from glucocorticoid-treated birds were incubated with chicken red blood cell (cRBC) nuclei. The thymocyte nuclear protein extract contained an endonuclease activity that degraded chromatin at internucleosomal sites and generated an amplified population of hypodiploid cRBC nuclei, similar to that detected in vivo in glucocorticoid-treated thymocytes. These data demonstrate a clear correlation between the detection of apoptotic endonuclease activity generated in vivo, where endogenous thymocyte DNA was degraded, with an in vitro assay, where extracted thymocyte nuclear proteins demonstrated a similar type of nuclease activity when cRBC nuclei were employed as an exogenous chromatin substrate.

Original languageEnglish
Pages (from-to)263-276
Number of pages14
JournalDevelopmental and Comparative Immunology
Volume17
Issue number3
DOIs
Publication statusPublished - 1993
Externally publishedYes

Fingerprint

Thymocytes
Lymphocytes
Apoptosis
Nuclear Proteins
Cell Nucleus
Glucocorticoids
Chickens
Erythrocytes
Endonucleases
Chromatin
Birds
In Vitro Techniques
DNA Cleavage
Agar Gel Electrophoresis
DNA
Confocal Microscopy
Dexamethasone
Population
Flow Cytometry

Keywords

  • Apoptosis
  • Confocal microscopy
  • DNA degradation
  • Endonuclease assay
  • Flow cytometry
  • Programmed cell death
  • Thymocytes

ASJC Scopus subject areas

  • Immunology
  • Developmental Biology

Cite this

Analysis of thymic lymphocyte apoptosis using in vitro techniques. / Machaca, Khaled; Compton, Mark M.

In: Developmental and Comparative Immunology, Vol. 17, No. 3, 1993, p. 263-276.

Research output: Contribution to journalArticle

@article{a0372532ebdb43e0b8ca471c1622dc30,
title = "Analysis of thymic lymphocyte apoptosis using in vitro techniques",
abstract = "Thymocyte apoptosis was analyzed in 4-week-old chicks that were treated with dexamethasone. Glucocorticoid treatment of birds resulted in the internucleosomal cleavage of thymocyte DNA as detected by agarose gel electrophoresis and the generation of a population of thymocyte nuclei with a hypodiploid content of cellular DNA when analyzed by flow cytometry. Visualization of apoptotic thymocytes at the ultrastructural level or via confocal microscopy revealed the typical morphological characteristics of cells undergoing apoptosis. Similar techniques were employed to further analyze apoptosis using an in vitro approach where extracts of thymocyte nuclear proteins from glucocorticoid-treated birds were incubated with chicken red blood cell (cRBC) nuclei. The thymocyte nuclear protein extract contained an endonuclease activity that degraded chromatin at internucleosomal sites and generated an amplified population of hypodiploid cRBC nuclei, similar to that detected in vivo in glucocorticoid-treated thymocytes. These data demonstrate a clear correlation between the detection of apoptotic endonuclease activity generated in vivo, where endogenous thymocyte DNA was degraded, with an in vitro assay, where extracted thymocyte nuclear proteins demonstrated a similar type of nuclease activity when cRBC nuclei were employed as an exogenous chromatin substrate.",
keywords = "Apoptosis, Confocal microscopy, DNA degradation, Endonuclease assay, Flow cytometry, Programmed cell death, Thymocytes",
author = "Khaled Machaca and Compton, {Mark M.}",
year = "1993",
doi = "10.1016/0145-305X(93)90045-R",
language = "English",
volume = "17",
pages = "263--276",
journal = "Developmental and Comparative Immunology",
issn = "0145-305X",
publisher = "Elsevier Limited",
number = "3",

}

TY - JOUR

T1 - Analysis of thymic lymphocyte apoptosis using in vitro techniques

AU - Machaca, Khaled

AU - Compton, Mark M.

PY - 1993

Y1 - 1993

N2 - Thymocyte apoptosis was analyzed in 4-week-old chicks that were treated with dexamethasone. Glucocorticoid treatment of birds resulted in the internucleosomal cleavage of thymocyte DNA as detected by agarose gel electrophoresis and the generation of a population of thymocyte nuclei with a hypodiploid content of cellular DNA when analyzed by flow cytometry. Visualization of apoptotic thymocytes at the ultrastructural level or via confocal microscopy revealed the typical morphological characteristics of cells undergoing apoptosis. Similar techniques were employed to further analyze apoptosis using an in vitro approach where extracts of thymocyte nuclear proteins from glucocorticoid-treated birds were incubated with chicken red blood cell (cRBC) nuclei. The thymocyte nuclear protein extract contained an endonuclease activity that degraded chromatin at internucleosomal sites and generated an amplified population of hypodiploid cRBC nuclei, similar to that detected in vivo in glucocorticoid-treated thymocytes. These data demonstrate a clear correlation between the detection of apoptotic endonuclease activity generated in vivo, where endogenous thymocyte DNA was degraded, with an in vitro assay, where extracted thymocyte nuclear proteins demonstrated a similar type of nuclease activity when cRBC nuclei were employed as an exogenous chromatin substrate.

AB - Thymocyte apoptosis was analyzed in 4-week-old chicks that were treated with dexamethasone. Glucocorticoid treatment of birds resulted in the internucleosomal cleavage of thymocyte DNA as detected by agarose gel electrophoresis and the generation of a population of thymocyte nuclei with a hypodiploid content of cellular DNA when analyzed by flow cytometry. Visualization of apoptotic thymocytes at the ultrastructural level or via confocal microscopy revealed the typical morphological characteristics of cells undergoing apoptosis. Similar techniques were employed to further analyze apoptosis using an in vitro approach where extracts of thymocyte nuclear proteins from glucocorticoid-treated birds were incubated with chicken red blood cell (cRBC) nuclei. The thymocyte nuclear protein extract contained an endonuclease activity that degraded chromatin at internucleosomal sites and generated an amplified population of hypodiploid cRBC nuclei, similar to that detected in vivo in glucocorticoid-treated thymocytes. These data demonstrate a clear correlation between the detection of apoptotic endonuclease activity generated in vivo, where endogenous thymocyte DNA was degraded, with an in vitro assay, where extracted thymocyte nuclear proteins demonstrated a similar type of nuclease activity when cRBC nuclei were employed as an exogenous chromatin substrate.

KW - Apoptosis

KW - Confocal microscopy

KW - DNA degradation

KW - Endonuclease assay

KW - Flow cytometry

KW - Programmed cell death

KW - Thymocytes

UR - http://www.scopus.com/inward/record.url?scp=0027216164&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027216164&partnerID=8YFLogxK

U2 - 10.1016/0145-305X(93)90045-R

DO - 10.1016/0145-305X(93)90045-R

M3 - Article

VL - 17

SP - 263

EP - 276

JO - Developmental and Comparative Immunology

JF - Developmental and Comparative Immunology

SN - 0145-305X

IS - 3

ER -