Detection by flow cytometry

Patel, A. K., Hallett, M. B., Campbell, A. K. (1987). Threshold responses in production of reactive oxygen metabolites in individual neutrophils detected by flow cytometry and microfluorimetry. Biochem. J. 248, 173-80. 

Membrane asymmetry changes can be detected by flow cytometry using a fluorescent marker (e.g. fluorescein, FITC) conjugated to annexin V, a protein that has high affinity for phosphatidylserine. When using a fluorescence microscope, this technique can be quantitative if a hemocyt-ometer is used. 

Figure 23.4. Schematic of induction of HTLV-I specific CD8+ T cells responses associated with immunopathogenesis of HAM/TSP. Quantitative PCR (DNA Taqman) demonstrates high HTLV-I proviral loads in HAM/TSP patients that are directly proportional to increased HTLV-I mRNA expression. In quantitative RT-PCR (RNA Taqman) analyses, elevated HTLV-I mRNA expression leads to increases of HTLV-I protein expression. This protein can be detected by flow cytometry. HTLV-I proteins (e.g.. Tax) can be processed into immunodominant peptides such as Taxi 1-19 peptide and presented on the cell surface of infected cells in context with MHC class I molecules. HTLV-I Taxi 1-19 peptide strongly binds to HLA-A 0201 molecules and stimulates a vims-specific CD8-I- T cells response. This Taxi 1-19/HLA-A 0201 complex can be detected with a TCR-like antibody, while the frequency of virus-specific CD8-I- T cells can be determined by HLA-A 0201/Taxl 1-19 tetramers. These antigen-specific responses are expanded in the CSF of HAM/TSP patients and may contribute to disease progression by recognition of HTLV-I-processed antigens in the CNS associated with lysis of HTLV-I-infected inflammatory cells, HTLV-I-infected glial cells, and/or through induction of proinflammatory cytokines and chemokines.

For intracellular cytokine detection by flow cytometry, PBMCs are often used, but to have a more specific analysis, it may be necessary to use cells from other biological fluids (e.g., synovial fluid, cerebrospinal fluid, and bronchoalveo-lar lavage fluid) or to separate cells according to functional characteristics or expression of membrane antigens (e.g., CD3, CD4, CDS, and CD56). 

Shedding does not always lead to diminished cell surface receptor. IL-4 and IL-10 are two cytokines with antiinflammatory effects on several cell types. IL-4 alone has no effect on surface TNF-R density but does induce shedding in rheumatoid synovial fibroblasts (Taylor, 1994), indicating that receptors are rapidly replaced. In cultures of peripheral blood mono-nucleated cells, 2-hr lL-10 treatment reduces surface TNF-R expression as detected by flow cytometry, but enhanced reexpression of TNF-Rs occurs by 48 hr (Leeuwenberg et al., 1994). Interestingly, IL-10 activates TNF-Rl and TNF-R2 shedding from these cells, whereas IL-4 has no such effect (Leeuwenberg et al., 1994). 

Petriz, J. Tugues, D. Garcia-Lopez, J. Relevance of forward scatter and side scatter in aneuploidy detection by flow cytometry. J. Eur. Soc. Anal. Cell. Pathol. 1996,... 

Hematologic A 10-year-old boy with acute leukemia developed an immune hemolytic anemia after taking co-trimoxazole for 3 days for Pneumocystis jirovecii prophylaxis [117 ]. A direct antiglobulin (Coombs ) test was strongly positive for IgG and C3, and an indirect antibody test was strongly positive in the presence of co-trimoxazole and trimethoprim, but not sulfamethoxazole alone trimethoprim-dependent erythrocyte antibodies were detected by flow cytometry. 

Telford, W.G., Moss, M.W., Morseman, J.P, AUnutt, F., 2001a. Cryptomonad algal phycobfliproteins asfluorochromes for extracellular and intracellular antigen detection by flow cytometry. Cytometry 44 (1), 16-23. 

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