Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry
| Autor: | Zuben E. Sauna, Tom E. Howard, Sandra C. Tseng, Gouri Shankar Pandey |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Article Subject
medicine.drug_class Gene Expression lcsh:Medicine CHO Cells Hemophilia A Monoclonal antibody Peripheral blood mononuclear cell General Biochemistry Genetics and Molecular Biology Cell Line Flow cytometry Mice 03 medical and health sciences 0302 clinical medicine Cricetinae hemic and lymphatic diseases medicine Animals Humans 030304 developmental biology 0303 health sciences Factor VIII General Immunology and Microbiology biology medicine.diagnostic_test lcsh:R Antibodies Monoclonal General Medicine Flow Cytometry Minimal residual disease Molecular biology Protein Structure Tertiary 3. Good health Blot HEK293 Cells Real-time polymerase chain reaction 030220 oncology & carcinogenesis Leukocytes Mononuclear biology.protein Antibody Intracellular Research Article |
| Zdroj: | BioMed Research International, Vol 2013 (2013) BioMed Research International |
| ISSN: | 2314-6133 |
| DOI: | 10.1155/2013/793502 |
| Popis: | Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs). An indirect intracellular staining (ICS) method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI) values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels. |
| Databáze: | OpenAIRE |
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