Flow cytometry analysis proteins

G. Georgiou Analysis of Large Libraries of Protein Mutants Using Flow Cytometry. Adv. Protein Chem. 55, 293 (2000). 

Georgiou, G. Analysis of large libraries of protein mutants using flow cytometry. Adv Protein Chem (2000) 55 293-315. 

Mechanistic studies [35] showed combination treatments to inhibit cell proliferation via downregulation of cyclin D1 and induce apoptosis via activation of caspases-8 and -9 and downregulation of prosurvival proteins, FLIP and survivin [35]. Several stUhenes, related to resveratrol, have been synthesized and tested for their anticancer effect on HL-60 leukemia cell line, taking particular care of the cell cycle analysis. Figure 8.2 shows synthesis of stilbenes and its effects on cell cyde. A scheme of flow cytometry analysis of cell cycle is presented in Figure 8.3. The most potent compound was found to be (Z)-3,4, 5-trimethoxystilbene(I)... 

Figu re 4.8 Flow cytometry analysis of silica nanoparticles surface-modified with proteins. MPMS nanoparticles (a, c) and TEOS nanoparticles (b, d) modified with GFP (a, b) or phycoerythrin-conjugated streptavidin... 

Successful cell surface display of the protein can be verified by inducing gene expression via addition of anhydrotetracycline to the culture medium and immunofluorescence staining of the cells using an antibody directed against the protein to be displayed (Protocol 1). Analysis can be performed by fluorescence microscopy or flow cytometry. 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

Fig. 7.1. Unpublished results from McNally A and Bauer KD, reprinted with permission from Bauer KD and Jacobberger JW (1994). Analysis of intracellular proteins. Darzynkiewicz Z, et al (eds). Flow Cytometry, 2nd edition. San Diego Academic Press, pp 351-376.

ANALYSIS OF LARGE LIBRARIES OF PROTEIN MUTANTS USING FLOW CYTOMETRY... 

The analysis of libraries by flow cytometry requires special attention to be paid to the protein expression system. An ideal expression system for isolation of live cells by flow cytometry must fulfill the following criteria (Daugherty et al., 1999) ... 

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