Quantitative cytometry of MHC class I digestion from living cells

Giacomo Galati, Cinzia Arcelloni, Rita Paroni, Silvia Heltai, Patrizia Rovere, Claudio Rugarli, Angelo A. Manfredi

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Digestion of crude membrane preparations with papain releases the extracellular portion of major histocompatibility complex (MHC) class I molecules. MHC class I molecules are integral membrane glycoprotein complexes formed by the noncovalent association of 2 invariant molecules, the heavy chain and the β2-microglobulin (β2-m), to a wide array of peptides. The cleaved soluble moiety retains the antigenic properties of the intact membrane-bound complex. Here we show that MHC class I digestion may be carried out on living cells, and we quantitate the surface expression of MHC complexes by a combined cytometric/high performance liquid chromatographic (HPLC) approach. Papain digestion results in time- and dose-dependent disappearance of membrane MHC-associated-fluorescence as detected by FACS analysis with MHC-specific monoclonal antibodies (mAbs). β2-m and peptides became detectable by HPLC analysis and western blotting in the digestion buffer and were quantitated by comparison with purified standards. The cytometric assessment of the digestion allows one to simultaneously monitor efficacy and toxicity of the treatment. The procedure we describe allows to selectively retrieve by affinity chromatography MHC from the cell membrane, avoiding any contamination due to intracellular, 'immature' MHC molecules.

Original languageEnglish
Pages (from-to)77-83
Number of pages7
JournalCytometry
Volume27
Issue number1
DOIs
Publication statusPublished - Jan 1 1997

Fingerprint

Major Histocompatibility Complex
Digestion
Papain
Membranes
Peptides
Membrane Glycoproteins
Affinity Chromatography
Buffers
Fluorescence
Western Blotting
High Pressure Liquid Chromatography
Monoclonal Antibodies
Cell Membrane

Keywords

  • β-microglobulin
  • cytometry
  • HPLC
  • MHC molecules
  • papain
  • peptides

ASJC Scopus subject areas

  • Hematology
  • Cell Biology
  • Pathology and Forensic Medicine
  • Biophysics
  • Endocrinology

Cite this

Quantitative cytometry of MHC class I digestion from living cells. / Galati, Giacomo; Arcelloni, Cinzia; Paroni, Rita; Heltai, Silvia; Rovere, Patrizia; Rugarli, Claudio; Manfredi, Angelo A.

In: Cytometry, Vol. 27, No. 1, 01.01.1997, p. 77-83.

Research output: Contribution to journalArticle

Galati, Giacomo ; Arcelloni, Cinzia ; Paroni, Rita ; Heltai, Silvia ; Rovere, Patrizia ; Rugarli, Claudio ; Manfredi, Angelo A. / Quantitative cytometry of MHC class I digestion from living cells. In: Cytometry. 1997 ; Vol. 27, No. 1. pp. 77-83.
@article{5405b98236d141e7b1cdefda476811c7,
title = "Quantitative cytometry of MHC class I digestion from living cells",
abstract = "Digestion of crude membrane preparations with papain releases the extracellular portion of major histocompatibility complex (MHC) class I molecules. MHC class I molecules are integral membrane glycoprotein complexes formed by the noncovalent association of 2 invariant molecules, the heavy chain and the β2-microglobulin (β2-m), to a wide array of peptides. The cleaved soluble moiety retains the antigenic properties of the intact membrane-bound complex. Here we show that MHC class I digestion may be carried out on living cells, and we quantitate the surface expression of MHC complexes by a combined cytometric/high performance liquid chromatographic (HPLC) approach. Papain digestion results in time- and dose-dependent disappearance of membrane MHC-associated-fluorescence as detected by FACS analysis with MHC-specific monoclonal antibodies (mAbs). β2-m and peptides became detectable by HPLC analysis and western blotting in the digestion buffer and were quantitated by comparison with purified standards. The cytometric assessment of the digestion allows one to simultaneously monitor efficacy and toxicity of the treatment. The procedure we describe allows to selectively retrieve by affinity chromatography MHC from the cell membrane, avoiding any contamination due to intracellular, 'immature' MHC molecules.",
keywords = "β-microglobulin, cytometry, HPLC, MHC molecules, papain, peptides",
author = "Giacomo Galati and Cinzia Arcelloni and Rita Paroni and Silvia Heltai and Patrizia Rovere and Claudio Rugarli and Manfredi, {Angelo A.}",
year = "1997",
month = "1",
day = "1",
doi = "10.1002/(SICI)1097-0320(19970101)27:1<77::AID-CYTO10>3.0.CO;2-P",
language = "English",
volume = "27",
pages = "77--83",
journal = "Cytometry Part B - Clinical Cytometry",
issn = "1552-4949",
publisher = "Wiley-Liss Inc.",
number = "1",

}

TY - JOUR

T1 - Quantitative cytometry of MHC class I digestion from living cells

AU - Galati, Giacomo

AU - Arcelloni, Cinzia

AU - Paroni, Rita

AU - Heltai, Silvia

AU - Rovere, Patrizia

AU - Rugarli, Claudio

AU - Manfredi, Angelo A.

PY - 1997/1/1

Y1 - 1997/1/1

N2 - Digestion of crude membrane preparations with papain releases the extracellular portion of major histocompatibility complex (MHC) class I molecules. MHC class I molecules are integral membrane glycoprotein complexes formed by the noncovalent association of 2 invariant molecules, the heavy chain and the β2-microglobulin (β2-m), to a wide array of peptides. The cleaved soluble moiety retains the antigenic properties of the intact membrane-bound complex. Here we show that MHC class I digestion may be carried out on living cells, and we quantitate the surface expression of MHC complexes by a combined cytometric/high performance liquid chromatographic (HPLC) approach. Papain digestion results in time- and dose-dependent disappearance of membrane MHC-associated-fluorescence as detected by FACS analysis with MHC-specific monoclonal antibodies (mAbs). β2-m and peptides became detectable by HPLC analysis and western blotting in the digestion buffer and were quantitated by comparison with purified standards. The cytometric assessment of the digestion allows one to simultaneously monitor efficacy and toxicity of the treatment. The procedure we describe allows to selectively retrieve by affinity chromatography MHC from the cell membrane, avoiding any contamination due to intracellular, 'immature' MHC molecules.

AB - Digestion of crude membrane preparations with papain releases the extracellular portion of major histocompatibility complex (MHC) class I molecules. MHC class I molecules are integral membrane glycoprotein complexes formed by the noncovalent association of 2 invariant molecules, the heavy chain and the β2-microglobulin (β2-m), to a wide array of peptides. The cleaved soluble moiety retains the antigenic properties of the intact membrane-bound complex. Here we show that MHC class I digestion may be carried out on living cells, and we quantitate the surface expression of MHC complexes by a combined cytometric/high performance liquid chromatographic (HPLC) approach. Papain digestion results in time- and dose-dependent disappearance of membrane MHC-associated-fluorescence as detected by FACS analysis with MHC-specific monoclonal antibodies (mAbs). β2-m and peptides became detectable by HPLC analysis and western blotting in the digestion buffer and were quantitated by comparison with purified standards. The cytometric assessment of the digestion allows one to simultaneously monitor efficacy and toxicity of the treatment. The procedure we describe allows to selectively retrieve by affinity chromatography MHC from the cell membrane, avoiding any contamination due to intracellular, 'immature' MHC molecules.

KW - β-microglobulin

KW - cytometry

KW - HPLC

KW - MHC molecules

KW - papain

KW - peptides

UR - http://www.scopus.com/inward/record.url?scp=0031027763&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031027763&partnerID=8YFLogxK

U2 - 10.1002/(SICI)1097-0320(19970101)27:1<77::AID-CYTO10>3.0.CO;2-P

DO - 10.1002/(SICI)1097-0320(19970101)27:1<77::AID-CYTO10>3.0.CO;2-P

M3 - Article

C2 - 9000588

AN - SCOPUS:0031027763

VL - 27

SP - 77

EP - 83

JO - Cytometry Part B - Clinical Cytometry

JF - Cytometry Part B - Clinical Cytometry

SN - 1552-4949

IS - 1

ER -