Technique cell kinetics

Cell kinetics is defined as the measurement of time parameters m biological systems. Traditionally, this has involved the use of radioactive precursors of DNA, such as tritiated thymidine (3HTdR), and autoradiography to detect their incorporation into DNA. This technique has provided detailed knowledge of cell kinetics in both in vitro and in vivo experimental systems. The technique, however, is time consuming and arduous and is not readily applicable to human tumor research because of ethical problems involved in incorporation of a radioisotope into DNA. 

The development of monoclonal antibodies which recognize halogenated pyrimidines such as 5-bromo-2-deoxyuridine (BrdU) incorporated into DNA (1) and of flow cytometric (FCM) techniques to simultaneously measure BrdU uptake and total DNA content (2) have led to a renaissance in cell kinetic studies. The speed and quantitative power of the flow cytometer, m conjunction with the specificity and sensitivity of monoclonal antibody techniques, provide the basis for the adoption and success of the BrdU technique in experimental and clinical investigations. 

The BrdU/FCM technique offers several advantages over 3HTdR/autorad-lography method in many cell kinetic studies. 

If a certain process can produce a product, it is important to know how fast the process can take place. Kinetics deals with rate of a reaction and how it is affected by various chemical and physical conditions. This is where the expertise of chemical engineers familiar with chemical kinetics and reactor design plays a major role. Similar techniques can be employed to deal with enzyme or cell kinetics. To design an effective bioreactor... 

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.

A comparison of the outcomes of this approach to single-cell protein synthesis kinetics determination with the alternative method which has been widely used in many previous studies of cell cycle and cell kinetic behavior of many organisms is instructive. In the latter methods, synchronous culture or an equivalent experimental technique are used to make measurements of the time variation of protein content or some other cellular variable versus time as the cell grows from a newborn daughter cell to a mature and dividing mother cell. Then, by estimating the slope of... 

Each of these cell kinetic parameters can be measured by different techniques although some, like 6, are difficult to quantitate and are usually extrapolated from other measurements. The BrdUrd/DNA staining technique can provide quantitative information on cell cycle phases and their duration, Tpot, and growth fraction. 

The development of antibodies to the halogenated pyrimidines combined with the attributes of FCM have led to a revolution in the study of cell kinetics and a resurgence of interest in their clinical apphcation. The techniques are reproducible and relatively straightforward and can be tailored to individual experimental requirements. 

Direct quantitation of receptor concentrations and dmg—receptor interactions is possible by a variety of techniques, including fluorescence, nmr, and radioligand binding. The last is particularly versatile and has been appHed both to sophisticated receptor quantitation and to dmg screening and discovery protocols (50,51). The use of high specific activity, frequendy pH]- or p lj-labeled, dmgs bound to cmde or purified cellular materials, to whole cells, or to tissue shces, permits the determination not only of dmg—receptor saturation curves, but also of the receptor number, dmg affinity, and association and dissociation kinetics either direcdy or by competition. Complete theoretical and experimental details are available (50,51). 

It is necessary to estabUsh a criterion for microbial death when considering a sterilization process. With respect to the individual cell, the irreversible cessation of all vital functions such as growth, reproduction, and in the case of vimses, inabiUty to attach and infect, is a most suitable criterion. On a practical level, it is necessary to estabUsh test criteria that permit a conclusion without having to observe individual microbial cells. The failure to reproduce in a suitable medium after incubation at optimum conditions for some acceptable time period is traditionally accepted as satisfactory proof of microbial death and, consequentiy, stetihty. The appHcation of such a testing method is, for practical purposes, however, not considered possible. The cultured article caimot be retrieved for subsequent use and the size of many items totally precludes practical culturing techniques. In order to design acceptable test procedures, the kinetics and thermodynamics of the sterilization process must be understood. 

Electrochemical systems are found in a number of industrial processes. In addition to the subsequent discussions of electrosynthesis, electrochemical techniques are used to measure transport and kinetic properties of systems (see Electroanalyticaltechniques) to provide energy (see Batteries Euel cells) and to produce materials (see Electroplating). Electrochemistry can also play a destmctive role (see Corrosion and corrosion control). The fundamentals necessary to analyze most electrochemical systems have been presented. More details of the fundamentals of electrochemistry are contained in the general references. 

Bastenie and Zylberszac, in a general article on the former subject, point out that colchicine (1) brings into mitosis all cells which are in karyo-kinetic inuninence but which normally would slowly and successively reach mitosis, and (2) stops them at this stage. This has made possible a technique which picks out cell multiplication and can be used for detecting many types of hormonal stimulation, e.g., the action of follicular hormone and other oestrogens. ... 

Let us examine some batch results. In trials in which 5 mL of a dye solution was added by pipet (with pressure) to 10 mL of water in a 25-mL flask, which was shaken to mix (as determined visually), and the mixed solution was delivered into a 3-mL rectangular cuvette, it was found that = 3-5 s, 2-4 s, and /obs 3-5 s. This is characteristic of conventional batch operation. Simple modifications can reduce this dead time. Reaction vessels designed for photometric titrations - may be useful kinetic tools. For reactions that are followed spectrophotometrically this technique is valuable Make a flat button on the end of a 4-in. length of glass rod. Deliver 3 mL of reaction medium into the rectangular cuvette in the spectrophotometer cell compartment. Transfer 10-100 p.L of a reactant stock solution to the button on the rod. Lower this into the cuvette, mix the solution with a few rapid vertical movements of the rod, and begin recording the dead time will be 3-8 s. A commercial version of the stirrer is available. 

The techniques referred to above (Sects. 1—3) may be operated for a sample heated in a constant temperature environment or under conditions of programmed temperature change. Very similar equipment can often be used differences normally reside in the temperature control of the reactant cell. Non-isothermal measurements of mass loss are termed thermogravimetry (TG), absorption or evolution of heat is differential scanning calorimetry (DSC), and measurement of the temperature difference between the sample and an inert reference substance is termed differential thermal analysis (DTA). These techniques can be used singly [33,76,174] or in combination and may include provision for EGA. Applications of non-isothermal measurements have ranged from the rapid qualitative estimation of reaction temperature to the quantitative determination of kinetic parameters [175—177]. The evaluation of kinetic parameters from non-isothermal data is dealt with in detail in Chap. 3.6. 

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