Modified flow cytometry and cell-ELISA methodology to detect HLA class I antigen processing machinery components in cytoplasm and endoplasmic reticulum.
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.
Ogino
Takeshi
T
Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Wang
Xinhui
X
Ferrone
Soldano
S
D000818:Animals; D017951:Antigen Presentation / Q000502:physiology*; D017920:Antiporters / Q000172:deficiency; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D015395:Histocompatibility Antigens Class I / Q000032:analysis* / Q000502:physiology; D016591:Histocytological Preparation Techniques / Q000379:methods; D006801:Humans; D007136:Immunoglobulins / Q000172:deficiency; D008214:Lymphocytes / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D026901:Membrane Transport Proteins; D001613:beta 2-Microglobulin / Q000172:deficiency
D017920:Antiporters; D015395:Histocompatibility Antigens Class I; D007136:Immunoglobulins; D026901:Membrane Transport Proteins; D001613:beta 2-Microglobulin; C107762:tapasin