Flow cytometry intracellular proteins

Although many applications of flow cytometry involve the staining of cells for proteins expressed on the outer membrane, cells also have many proteins that are not displayed on their surface. With appropriate procedures, flow cytometry can provide a means to analyze these intracellular proteins. The outer cell membrane is impermeable to large molcules like antibodies however, if we intentionally fix cells to stabilize proteins and then disrupt the outer membrane, the cells can be stained with fluorochrome-conjugated monoclonal antibodies against intracellular proteins. After time to allow the antibodies to pass through the now-permeabilized membrane, the cells are washed to remove loosely bound antibodies and then are run through the flow cytometer to measure their fluorescence intensity. 

Fig. 7.3. A dot plot (on the left) showing the staining of cells from a human breast tumor for two intracellular proteins. Cytokeratin-positivity marks tumor cells in the suspension, and estrogen receptor positivity on these cells indicates superior prognosis. The plot on the right shows a correlation (in 27 breast tumors) between the intensity of estrogen receptor staining by flow cytometry and the level of estrogen receptor binding (by radioligand binding assay). Modified from Ian Brotherick et al. (1995).

Fixation Fixation is the process by which the protein of cells is denatured, or cross-linked, and preserved. Fixation in flow cytometry is used to inactivate hazardous biological material and also to preserve stained cells when there is not immediate access to a flow cytometer. Fixation is also important in preserving proteins before detergent permeabilization for intracellular staining. Formaldehyde is often the fixative of choice for flow cytometry because it preserves the forward and side scatter characteristics of cells (but does cause some increase in their autofluorescence). 

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 Unpublished results from McNally A and Bauer KD, reprinted with permission from Bauer KD and Jacobberger JW (1994).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.

Among the items that have been measured are vitality, intracellular pH, DNA and RNA content, and specific plasmids [77,408]. Besides nucleic acids [204], other intracellular components can also be analyzed, e.g. storage materials [2, 82,294], enzymes and protein content [6,338], or the cell size [60,61]. The physiological state can also be rapidly assessed [331]. Furthermore, this technique allows the separation of certain cells using a cell sorter, e.g. for strain improvement [28]. The flow cytometry technique has also been used in connection with molecular probes for identification and viability determination of microbial communities [98]. This application of viability estimation is becoming increasingly important [63, 136, 188, 454]. Unfortunately, the equipment is expensive and most of the measurements are tricky and laborious and not well designed for on-line application. 

Pollice, A A, McCoy, J. P, Jr., Shackney, S E, Smith, C A., Agarwal, J., Burholt, D R., Janocko, L. E, Hornicek, F J, Singh, S. G, and Hartsock, R. J. (1992) Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell-surface proteins, and intracellular proteins. Cytometry 13,432-444... 

In this contribution a population balance model of influenza A vims replication during vaccine production in Madin-Darby canine kidney (MDCK) cell cultures is developed. Differentiation on the population level is described by a degree of infection, which is proportional to the amount of intracellular viral proteins. This can be measured directly using flow cytometry. It is shown that the model shows reasonable agreement with experimental data, although not all details of the inner dynamics can be fully reproduced. 

Based on the experimental investigation of the infection status of cells by measuring immrmofluorescence of intracellular viral proteins with flow cytometry [3] mathematical models are required, which are able to describe distributed populations of cells with different degrees of infection. For this purpose, in the present paper an internal coordinate is introduced to quantify the degree of infection and the previous approach by Mohler et al. [4] is extended accordingly. 

Krutzik PO, Nolan GP. Intracellular phospho-protein staining techniques for flow cytometry monitoring single cell signahng events. Cytometry A 2003 55 61-70. 

Fluorescent derivatives of carbohydrate-binding proteins have been used to detect cell-surface and intracellular glycoconjugates by microscopy and flow cytometry, to localize glycoproteins in gels and on protein blots, to precipitate glycoproteins in solution and to cause agglutination of specific cell types. 

Differentiation Flow cytometry, cell surface, and intracellular staining ELISA for secreted proteins... 

Cell activation can lead to differentiation of certain cells into more mature cells that exhibit different structural and functional form. For example, immature hematopoietic cells can be stimulated by some proteins to mature, by changing cell morphology and by inducing expression of different intracellular and surface markers. Expression of differentiation markers can be detected and monitored by flow cytometry using specific antibodies against cell surface or intracellular markers. 

Intracellular proteins associated terminal deoxynucleotidyl-transferase (TdT) Flow-cytometry based bead assay nick end labeling of broken ends of the double stranded DNA Detects proteins from cells associated with different stages of Fluorescence... 

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