Fluorescence fluorescein

Table I describes several of the fluorescent assays that have been used in our lab to study neutrophil activation. Fluorescein-labeled W-formylhexapeptide (FLPEP) has been used to characterize the ki- netics of ligand binding, dissociation, and internalization at 37°C (7,8). FLPEP is added to a suspension of cells, then receptor-bound and free FLPEP in solution are distinguished by adding antibody to fluorescein. This is a high-affinity antibody which binds free FLPEP within 1 s hut does not bind cell-bound FLPEP. When it binds the FLPEP, it quenches the fluorescein fluorescence. Hence the residual fluorescence after antibody addition represents FLPEP that is bound to the cell. Nonspecific binding is determined in cell suspensions that contain an excess of nonfluorescent peptide.

Keywords Benzoxadiazoles Coumarins Fluorescein Fluorescent probes Rhodamine... 

Add 500 J,L of crystal violet solution to the reaction mixture, and analyze another 10,000 cells. Crystal violet quenches extracellular fluorescein fluorescence of attached, uningested particles (18). The histogram profile in the presence of crysta 1 violet represents the fluorescence of internalized particles (see Note 8). 

When the photodetector for fluorescein fluorescence is properly set on the flow cytometer, the dye-loaded, unstimulated cells (Fig. 3A, shaded curve) should be visible on scale with the dye-loaded, particle-stimulated cells (Fig. 3A, open curve) (see Note 2). 

Figure 6. Elution profile obtained on examination of the gel permeation behavior of 14C acetylated S. aureus Factor III. Cytochrome-c and fluorescein labeled E. coli HPr were included in the applied sample as internal standards. Absorbance at 410 nm (cytochrome-c), fluorescein fluorescence (HPr), and radioactivity (Factor III) are plotted vs. fraction number. Inset shows the elution profiles for the external (blue dextran) and internal (fiuoroglycine) column markers. Data in this figure are from unpublished work (20).

Figure 7. Elution profile for a sample of myoglobin which was partially cleaved with cyanogen bromide and labeled with fluorescein isothiocyanate. Absorbance at 280 fim, (broken line) and fluorescein fluorescence, 493-nm excitation, and 530-nm emission, (solid line) are plotted vs. fraction number. The identities of components A, B, C, D, and E were inferred on the basis of their Mav s (see...

Oxygen radical absorbency capacity (ORAC) method presumes registration of substrate (B-phycoerythrin or fluorescein) fluorescence [43,44] after reaction with AOC in the sample to be compared with reference template. Trolox is used as a standard to determine per-oxyl radicals and gallic acid to determine hydroxyl radicals. 

Fig. 1.1. The optical path of a fluorescence microscope. In this example, the filters and mirrors are set for detection of fluorescein fluorescence. From Alberts et al.

The third problem that needs to be controlled is that of spectral cross-over and the possibility of incorrect instrument compensation. As an example of a case in which controls for nonspecific staining, autofluorescence, and compensation are all critical, let us look at the staining of B lymphocytes for the CD5 marker present with only low density on their surface. As well as the problems created by nonspecific staining and by autofluorescence, the problem of spectral cross-over between fluorescein and phycoerythrin can particularly confuse the interpretation of results from this kind of experiment. Look at Figure 6.5. What we are interested in is the number of B lymphocytes that possess the CD5 surface antigen. These cells will appear in quadrant 2 of a contour plot of fluorescein fluorescence... 

Figure 34 Stem-Volmer plots for the quenching of fluorescein fluorescence by rhodamine B in (a) water, CTAB, DDAB, CTAB-ZrP, and (b) water, DDAB-ZrP. (From Ref. 69. Copyright 2000 Elsevier Publishers.)...

Experiment 1. Studies on the Accessibility of I- to a Fluorophore Quenching of Fluorescein Fluorescence with Kl... 

Protein fusions displayed per cell (Fluorescein fluorescence)... 

Either electronically or through the computer software, correct for spectral overlap between the ports (for example, for fluorescein fluorescence recorded in the PE channel). 

Homogeneous Fluorophore Fluorescein Fluorescence polarization Fluorescence quenching Fluorescent energy transfer... 

FIGURE 20.8 Calibration of the pH measurement. R, the ratio of tetramethylrhodamine and fluorescein fluorescence, versus the pH for liposomes constructed with internal pH values ranging from 3 to 7. The average ratio and standard deviation is shown with a marker and bars at 7 pH values. The calibration curve represents a quadratic model and was determined by multiple regression analysis. (Reprinted from Chen, Y. and Arriaga, E. A., Anal. Chem., 78, 821, 2006. Copyright 2006. With permission from American Chemical Society.)... 

Make microelectrodes—we use 1.2-mm diameter, thin-walled aluminosilicate glass with internal filament (A-M Systems, Everett, WA). Electrode tip should have a nice constant taper and be fine but not wispy. When back-filled with dextran and 1M potassium chloride, they should have a resistance of between 50 and 150MQ. In practice, the precise electrode resistance does not matter if the electrode penetrates a cell, records a stable membrane potential, and passes sufficient dye, then it is a good electrode (see Notes 7 and 8). Electrodes should first be back-fiUed with approx 0.5 0L of fluorescent dextran (lOOmg/mL in distilled water. Molecular Probes cat. no. D-3308 and D-3306 for tetramethylrhodamine and fluorescein fluorescence, respectively) and then with a little 1M potassium chloride. Don t worry about air bubbles. The capillary action of the internal filament will deal with them. 

Mix up a small ahquot of fluorescent dextran. Use Molecular Probes product numbers D-3308 and D-3306 for tetramethylrhodamine and fluorescein fluorescence, respectively. The author keeps a small frozen stock of dextran made up at a concentration of lOOmg/mL in distilled water from which he transfer a very small drop onto a Sylgard surface. As the water evaporates off, the dextran becomes sticky and is easily picked up on to the tip of a stainless-steel or tungsten micropin or onto the tips of watchmaker s forceps. Simply add more water to the drop of dextran if it dries out too much. 

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