Antigens by Flow Cytometry

Fig. 1. Scheme showing the basic strategies available for the detection of nuclear antigens by flow cytometry. PF, paraformaldehyde EtOH, ethanol MeOH, methanol. 

Millard, I., Degrave, E., Philippe, M., and Gala, J.-L. 1998. Detection of intracellular antigens by flow cytometry Comparison of two chemical methods and microwave heating. Clin. Chem. 44 2320-2330. 

C., and Janossy, G. 1994. Detection of membrane and intracellular antigens by flow cytometry following ORTHO PermeaFix fixation. Leukemia 8 672-676. 

In another experimental setting in vitro data on the cell surface antigen expression were obtained for PBMCs isolated from chronic lymphocytic leukemia (CLL) patient blood samples and cultured in the presence of DIMS (according to standard procedures). Cultured cells were collected and stained with the corresponding antibodies for detection of the expression of cell surface antigens. Subsequendy they were analyzed by flow cytometry. 

Positive selection is not recommended as these procedures may induce signaling pathways in CLL cells due to antibody/antigen interactions. Purity of CLL cells is assessed by flow cytometry using anti-CD19, anti-CD5, and matching isotype control... 

This will depend on the affinity of the MAb and the density of the antigen on the cells, but a final concentration of 5 pg/mL will be adequate for most commercially available MAbs If necessary, the concentration can be determined empirically by using serial dilutions of MAb and analyzing uptake by flow cytometry (see Chapter 33)... 

Aptamers attached to a fluorescent reporter are useful for quantification of a cell surface antigen on living cells by flow cytometry (see references [21, 38]). 

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). 

CD3 obtained with lot no. 1 antibody-beads was consistently closer to the %CD3 measured by flow cytometry. This was consistent with CD3/CD3-PE (PE = R-phycoerythrin) competitive binding experiments performed by flow cytometry, which showed that lot no. 1 CD3 antibody had a higher affinity for its antigenic receptors on T-cells, A assoc = b9 x 10 than lot no. 2 CD3 antibody, which had A assoc = 1.1. 5 x The binding constant of CD3-PE for the same... 

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