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Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry
Published on: September 28, 2016
Modified flow cytometry and cell-ELISA methodology to detect HLA class I antigen processing machinery components in
Takeshi Ogino1, Xinhui Wang, Soldano Ferrone
1Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Insights
New flow cytometry and cell-ELISA methods detect intracellular and endoplasmic reticulum (ER) antigens. These techniques enable sensitive analysis of HLA class I antigen processing machinery components, crucial for immune system recognition.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Flow cytometry and ELISA are standard for cell surface antigen analysis.
- Detecting intracellular and endoplasmic reticulum (ER) antigens is challenging with current methods.
- Accurate analysis of HLA class I antigen processing machinery is vital for understanding immune responses.
Purpose of the Study:
- To develop and validate modified flow cytometry and cell-ELISA techniques for detecting cytoplasmic and ER-located antigens.
- To assess the sensitivity, simplicity, and reproducibility of these modified methods.
- To enable comprehensive analysis of HLA class I antigen processing machinery components.
Main Methods:
- Cells were sequentially fixed with paraformaldehyde, microwave-treated, saponin-permeabilized, and incubated with monoclonal antibodies (mAbs).
- Flow cytometry and cell-ELISA were employed to detect intracytoplasmic (LMP10) and ER luminal (calreticulin, tapasin) markers.
- Ten human cell lines were tested using specific mAbs against HLA class I antigen processing machinery components.
Main Results:
- Modified flow cytometry and cell-ELISA successfully detected cytoplasmic and ER antigens.
- The methods demonstrated sensitivity, simplicity, and reproducibility.
- Results from the modified techniques showed significant correlation across 10 human cell lines.
Conclusions:
- The developed flow cytometry and cell-ELISA methods are effective for analyzing HLA class I antigen processing machinery components.
- These techniques facilitate the study of antigen processing in both physiological and pathological conditions.
- Improved understanding of antigen processing machinery expression can enhance characterization of cellular immune recognition.
Abstract:
Flow cytometry and cell-enzyme linked immunosorbent assay (ELISA) are useful techniques for the quantitative analysis of cell surface antigen expression. Furthermore, flow cytometry can detect intracellular markers in cells permeabilized to facilitate the intracellular penetration of antibodies. However, to the best of our knowledge, neither method has been used to detect antigens located in the endoplasmic reticulum (ER) of cells. This limitation has a negative impact on the analysis of the expression of HLA class I antigen processing machinery components in cells. Therefore in this study, we show that markers located in cytoplasm and ER can be detected by flow cytometry and cell-ELISA in cells sequentially fixed with paraformaldehyde, heated in a microwave oven, permeabilized with saponin and reacted with monoclonal antibodies (mAb). Utilizing LMP10 as an intracytoplasmic marker and calreticulin and tapasin as ER luminal markers, we show that the modified flow cytometry and cell-ELISA are sensitive, simple and reproducible methods to detect HLA class I antigen processing machinery components in cells. Furthermore, testing of 10 human cell lines with HLA class I antigen processing machinery component-specific mAb has shown that the results obtained with the modified flow cytometry and cell-ELISA are significantly correlated. These results altogether indicate that the modified flow cytometry and cell-ELISA methods we have described will facilitate the analysis of the expression of HLA class I antigen processing machinery components in cells under physiological and pathological conditions. The resulting information will contribute to the characterization of the effect of changes in the expression of antigen processing machinery components on the recognition of cells by the host's immune system.
