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 journalArticlepeer-review

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

Keywords

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

ASJC Scopus subject areas

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

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