Cell fluorescence measurement

The measurements by LSC are rapid and with optimal cell density on the slide up to 5000 cells can be measured per min. The accuracy and sensitivity of cell fluorescence measurements by LSC are similar as with advanced... 

Figure 2. The binding and dissociation of FLPEP and receptor on intact neutrophils at 37 C The data are plotted as the specific binding of FLPEP (pmoles/10 cells) on a log plot versus time. Experimental details 10 cells/mL were exposed at time 0 to 1 nAf FLPEP. At 15, 30, 60, or 120 s, antibody to fluorescein is added to each sample. Fluorescence is monitored continuously during the additions. The data ate derived from a point-by-point comparison of the fluorescence measured under conditions of receptor binding and receptor blockade. Data are representative of observations in more than 10 separate experiments. (Reproduced with permission from reference 22. Copyright 19S7 Journal of Biological Chemistry.)...

Fig. 3. Flow cytometry analysis. CellQuest software. The fluorescence of 50,000 cells is measured.

Figure 4.7 Changes in intraceiiuiar calcium in cultured rat ventricular myocytes exposed to oxidant stress. Calcium was measured using the fluorescent probe Fura>2. The ratio of the Fura-2 fluorescence measured at 340 and 380 nm excitation is shown and this is proportional to the intracellular calcium concentration. The fast-speed traces shown (note the 3.5 s time-scale) were recorded after various durations of oxidant stress. Myocytes under control conditions (before t = 0) show spontaneous calcium transients. These transients decreased in frequency with oxidant stress until cells failed to show spontaneous activity but continued to maintain a low intracellular calcium.

Fluorescence-based methods do not directly measure ionic current but, rather, measure either membrane-potential-dependent or ion-concentration-dependent changes of fluorescence signals (from fluorescent dyes loaded into the cytosol or cell membrane) as a result of ionic flux. Because fluorescence-based methods give robust and homogeneous cell population measurement, these assays are relatively easy to set up and achieve high throughput. 

Fluorescence measurement using this probe does not require a fluorescence quencher or washing process to suppress the fluorescence emission from nonbinding probes and nonspecific binding probes, which would be advantageous for the detection of mRNAs with poly(A) tracts in cells. 

The calcein-AM assay [82-84] and cytotoxicity assays (e.g., performed with doxorubicin) [77, 78] are both basically competition assays. The accumulation of a primary substrate (e.g., calcein-AM or doxorubicin) in the cytosol of living cells is measured after addition of a second substrate (also called modifier or reverser) that reduces the efflux of the primary substrate. In the case of the calcein-AM assay, the primary substrate, calcein-AM, is hydrolyzed as soon as it reaches the cytosol, and the highly fluorescent hydrolysis product (calcein) can be determined using fluorescence spectroscopy. The more effective the reversal agent, the stronger is the increase in calcein fluorescence. Data can be quantified in terms of inhibitory constants, IQ, of the reversal agent. 

As with the UV absorption detector, the sample compartment consists of a special cell for measuring a flowing, rather than static, solution. The fluorescence detector thus individually measures the fluorescence intensities of the mixture components as they elute from the column (see Figure 13.10). The electronic signal generated at the phototube is recorded on the chromatogram. 

A. C. Chao, J. A. Dix, M. C. Sellers, and A. S. Verkman, Fluorescence measurement of chloride transport in monolayer cultured cells. Mechanisms of chloride transport in fibroblasts, Biophys. J. 56, 1071-1081 (1989). 

In addition to the studies mentioned above, fluorescence measurements have also been applied to many other areas such as mixing time studies/25 32) mammalian cell culture/331 and immobilized cell cultures/34-361... 

Laser microbeams offer several advantages over other fluorescence excitation techniques. In spectrofluorometry, observations are often made on a population of cells in a cuvette, resulting in a combined signal that lacks information about individual cellular responses. In flow cytometry, many individual cells are measured, but there is no temporal resolution since each cell is observed only once, and there is no spatial resolution since the entire cell is illuminated as it passes through the laser beam (see Chapter 30). In conventional fluorescence microscopy, individual cells can be monitored over time, and information about the two-dimensional spatial distribution of fluorescence can be obtained. However, some samples may be more susceptible to photobleaching by the arc lamps used for excitation, and the temporal resolution is limited to video-rate data acquisition (30 frames/s) (see Chapter 14). 

Rufer N, Brummendorf TH, Kolvraa S et al. Telomere fluorescence measurements in granuloc34 es and T lymphoc34e subsets point to a high turnover of hematopoietic stem cells andmemoryT cells in early childhood. J.Exp.Med. 1999 190 157-167. 

This problem is overcome by the Bio View sensor, which offers the possibility to monitor the whole spectral range simultaneously, and by using suitable data analysis and mathematical methods like chemometric regression models 11061. Real-time fluorescence measurement can be used more effectively comparing time-consuming off-line methods. Partial least squares (PLS) calibration models were developed for simultaneous on-line prediction of the cell dry mass concentration (Fig. 5), product concentration (Fig. 6), and metabolite concentrations (e. g., acetic acid, not shown) from 2D spectra. 

Burchard, P., Bilger, W., and Weissenbock, G., Contribution of hydroxycinnamates and flavonoids to epidermal shielding of UV-A and UV-B radiation in developing rye primary leaves as assessed by ultraviolet-induced chlorophyll fluorescence measurements, Plant Cell Environ., 23, 1373, 2000. 

B. Fanget, O. Devos, and M. Draye, Correction of Inner Filter Effect in Mirror Coating Cells for Trace Level Fluorescence Measurements, Anal. Chem. 2003, 75, 2790. 

Figure 6. Instrumental schematic for vacuum UV photofragmentation-laser induced fluorescence measurement of ammonia SHGC, second harmonic generation crystal SFMC, sum frequency mixing crystal BS, beam splitter BD, beam dump TP, turning prism CL, cylindrical lens R, reflector TD, trigger diode OSC, oscillator cell AMP, amplifier cell BE, beam expander G, grating OC, output coupler M, mirror BC, beam combiner L, lens A, aperture PD, photodiode SC, sample cell RC, reference cell FP, filter pack SAM.PMT, sample cell photomultiplier REF.PMT, reference cell photomultiplier PP, additional photomultiplier port EX, exhaust and CGI, calibration gas inlet to flow line. (Reproduced with permission from reference 15. Copyright 1990 Optical Society of America.)...

For example, the oscillatory change in intracellular [Ca2+] shown above was observed in pancreatic insulin-secreting P cells responding to stimulation by the agonist carbamoylcholine. The free [Ca2+] was evaluated from fluorescence measurements using the Ca2+ indicator dye fura 2 (From Prentki et alss). Oscillations in [Ca2+] have been observed... 

The complexity of the data generated by multiparameter flow cytometric analysis means that the scatter and fluorescence measurements from individual cells are stored on the computer in the form of a list (list mode data). This means that the relationships between measurements on individual cells is preserved, and these relationships can be explored by effectively rerunning the analysis on the computer. 

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