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Quantitative cytometry of MHC class I digestion from living cells
G Galati1, C Arcelloni, R Paroni
1Laboratori di Immunologia dei Tumori e di Tecniche Separative, Istituto Scientifico H.S. Raffaele and Università di Milano, Milan, Italy.
Insights
Papain digestion of living cells releases soluble major histocompatibility complex (MHC) class I molecules. This method quantifies surface MHC expression and enables selective retrieval of intact MHC complexes.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Major histocompatibility complex (MHC) class I molecules are crucial for immune response.
- These molecules are integral membrane glycoproteins composed of a heavy chain and beta2-microglobulin (β2-m).
- The extracellular portion of MHC class I can be released by papain digestion, retaining antigenic properties.
Purpose of the Study:
- To investigate the feasibility of performing papain digestion on living cells for MHC class I analysis.
- To quantify cell surface MHC complex expression using a combined flow cytometry and HPLC approach.
- To develop a method for selective retrieval of cell membrane MHC molecules.
Main Methods:
- Papain digestion of living cells to release extracellular MHC class I molecules.
- Flow cytometry (FACS) with MHC-specific monoclonal antibodies (mAbs) to detect fluorescence changes.
- High-performance liquid chromatography (HPLC) and western blotting to detect and quantify released β2-m and peptides.
- Affinity chromatography for selective MHC retrieval.
Main Results:
- Papain digestion on living cells led to a time- and dose-dependent decrease in membrane MHC-associated fluorescence.
- β2-m and peptides were detected in the digestion buffer via HPLC and western blotting.
- Flow cytometry allowed simultaneous monitoring of digestion efficacy and potential toxicity.
- The procedure enabled selective isolation of MHC molecules from the cell membrane.
Conclusions:
- Papain digestion is effective on living cells for releasing extracellular MHC class I.
- The combined cytometric/HPLC approach accurately quantifies MHC surface expression and digestion efficiency.
- This method allows for the selective retrieval of intact MHC molecules from the cell surface, avoiding intracellular contaminants.
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 beta2-microglobulin (beta2-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). beta2-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.