Flow cytometry kinetics

There is a single assumption in these measurements--namely that the antibody only quenches free ligand. This has been demonstrated specifically by flow cytometry in experiments which show that there is no quenching of ligand on the cell (3). The kinetic analysis depends on rapid interaction of ligand and antibody, which in these experiments is essentially within the mixing time. 

Fig. 13.4 Cellular uptake kinetics of LDH-FITC as a function of concentration (A) and incubation time (B) in HOS cells. Cells were incubated with LDH-FITC for2h (A), concentration of LDH-FITC was 200pg/ml (B). The cellular uptake was quantified by flow cytometry.

Flow cytometry (FCM) is widely used for exploring mechanism of action of compounds that compromise proliferation since it is rapid, accurate and usable for any cellular context [5], In this chapter we want to point out technical and strategic aspects of use of FCM for cell cycle studies of a putative anticancer agent. As an example we used Edotecarin, a topi inhibitor, firstly evaluating proliferation outcome and classical DNA content analysis by propidium iodide, and then since the compound treatment produced cell cycle perturbation difficult to interprete, a two-parametric analysis by 5-bromo-deoxyuridine (BrdU) was applied for separating cell cycle phases. Moreover we put our efforts into identifing specific cell cycle arrest not easily demonstrable by previously described methods, through the use of in vitro kinetics ( pulse and chase ). Finally, in vivo assessment of efficacy and biomarkers modulation after treatment was analyzed. 

In our studies, we used flow cytometry to gain quantitative information in terms of uptake kinetics and extent of uptake inhibition after treatment with several inhibitors. 

Kinetic solubility This pragmatic approach starts with a concentrated compound solution in pure DM SO further diluted in a buffer medium. The amount of compound in solution is measured after a few minutes incubation either by recording its UV absorbance (with or without a chromatographic step) or precipitate formation using an optical method (turbidimetry, nephelometry or flow cytometry). This approach mimics the typical path of the compound in biochemical, cellular assays or in vivo animal models. Kinetic solubility usually serves as a quality filter prior to cell based assays (see paragraphs on solubility, permeability and cellular assays). 

BrdU/DNA flow cytometry offers flexibility and diversity in the study of cell kinetics from cells in culture to human tumors in vivo. The essence of the procedure is to pulse label with BrdU by a short-term incubation in vitro or by a single injection in vivo samples are then taken at time intervals thereafter and stained after fixation in ethanol. The cells are then stained with a monoclonal antibody against BrdU that can be either directly conjugated to a fluoro-chrome (usually fluorescein isothiocyanate [FITC]) or, alternatively, bound to a second antibody conjugated with FITC. The cells are then counterstained with propidium iodide (PI) to measure the DNA content and analyzed on the flow cytometer. The results are displayed as linear-red fluorescence on the x-axis vs linear or log-green fluorescence on they-axis. 

Wilson, G. D, McNally, N. J., Dische, S., Saunders, M. I., des Rochers, C, Lewis, A. A., and Bennett, M H. (1988) Measurement of cell kinetics in human tumours in vivo using bromodeoxyuridine incorporation and flow cytometry. Br J Cancer 58,423-431. 

Roe, R., Robins, R. A., Laxton, R R., and Baldwin, R W (1985) Kinetics of divalent monoclonal antibody binding to tumor cell surface antigens using flow cytometry—standardization and mathematical analysis Mol Immunol. 22, 11—21... 

Summary of Experimental Results of LRG Dissociation Kinetics Measured by Small Volume Rapid Mix Flow Cytometry... 

Wu, Y., Zwartz, G., Lopez, G. P., Sklar, L. A., and Buranda, T. (2005b). Small-volume rapid-mix device for subsecond kinetic analysis in flow cytometry. Cytometry Part A. 67(1), 37-44. 

Whereas the applications discussed so far in this book have dealt with ways to describe cells or nuclei that have been stopped in their tracks for analysis at a given moment, flow cytometry has also been used to follow the physiological function of cells in true kinetic analysis. As an example of this kind of analysis, we can discuss the use of a flow... 

Fig. 8.7. Reprinted (A,D) from Dean PN (1987). Data analysis in cell kinetics. Gray JW and Darzynkiewicz Z (eds). Techniques in Cell Cycle Analysis. Clifton, NJ Humana Press, pp 207-253 and (B,C) from Dean PN (1985). Methods of data analysis in flow cytometry. Van Dilla MA, et al. (eds). Flow Cytometry Instrumentation and Data Analysis. London Academic Press, pp 195-221.

Fig. 8.13. Reprinted with permission of Oxford University Press from McNally NJ and Wilson GD (1990). Measurement of tumour cell kinetics by the bromodeoxy-uridine method. Ormerod MG (ed). Flow Cytometry A Practical Approach. Oxford IRL, pp 87-104.

Ligand binding equilibria and dissociation kinetics can be readily determined by flow cytometry of whole cells (or, if desired, using proteins... 

Flow cytometry is well suited for the analysis of enzyme activity and kinetics at the single cell level (Watson and Dive, 1994). Flow cytometric assays for numerous enzymes including esterases, proteases, peroxidases, lipases, and oxidoreductases3 are available and are widely used in research and clinical practice. To date, flow cytometry has not been widely exploited as a screening tool for enzyme engineering purposes, but this is rapidly changing. 

Key Words Pulsed low electric fields Endocytosis Uptake kinetics Membrane internalization Albumin Lucifer Yellow Propidium iodide Adsorption Flow cytometry Confocal microscopy. 

Button, D. K., and Robertson, B. R. (1989). Kinetics of bacterial processes in natural aquatic systems based on biomass as determined by high-resolution flow-cytometry. Cytometry 10, 558—563. 

Fig. 4. Percent uptake of different iiposomai formuiations conjugated to Rtiodamine by human DCs measured through flow cytometry. Ceiis were incubated with Rhodamine-MSP-1, g-ioaded iiposomai formuiations and anaiyzed at different time intervals (0,15,30,60,120,180, and 360 min). The kinetics of uptake has been presented with mean fluorescence intensity (MFI) vs. counts by FACS analysis and percentage uptake at various time inten/als. Uptake of formulation on a per cell basis was quantified as fluorescence intensity per cell. Percentage of positive cells was determined as proportion ot cells with fluorescence intensity higher than 99% of cells of the control sample (cells incubated with unconjugated rhodamine alone). Flow cytometric analysis revealed that the percentage of rhodamine-positive DCs increased rapidly and reached a plateau after 16 h of incubation (means of three independent experiments). A steady increase in the uptake percentage (%) was recorded and a maximum cell-associated fluorescence was observed at 16 h for OPM-coated cationic liposomes. Flow cytometric analysis of DCs revealed that plain liposomes did not significantly enhance the antigen uptake by DCs compared with the uptake recorded for mannan-coated liposomes...

Yeast display uses the a-agglutinin receptor to display recombinant proteins on the surface of Saccharomyces cerevisiae. Yeast display of scFvs or Fabs allows the detection and selection by fluorescence-assisted flow cytometry or by magnetic sorting. In addition, flow cytometry can be used for kinetic characterization of antibody affinity (K ) as well as K ff and rates. ... 

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