Cell-associated fluorescence intensity

During chase incubation at 4°C, no significant change of the cell-associated fluorescence intensity should be detected over time. Because of the reduced incubation temperature, the metabolism of the cells is minimized, resulting in an inhibition of active uptake processes. Upon subsequent addition of monensin, the fluorescence emission signals should not be altered as well. In doing so, any direct influence of monensin on the quantum yield of the fluorescein label can be excluded [25],... 

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...

Fig. 4. Comparison of the concentration-dependent uptake of Ho33342 (1 h exposure) by three human cell lines as measured by flow cytometry. Ho33342-DNA associated fluorescence intensity measurements were corrected for the mean amount of DNA within a given cell population and related to a biological standard (i.e. HT29 cells exposed to 8.7 tiM Ho33342 for 1 h at 37 ). Symbols O hT29 MRC5CVI A AT5BIVA. Data points represent mean of at least two determinations (SE < 10%). (Data from [181 by permission of IRL Press)...

CFP-YFP donor-acceptor pair, YFP is several times brighter than CFP [62]. Lastly, for studying dynamic protein associations in plants, the presence of chlorophyll pigments in leaf and stem cells is an additional limitation. These pigments directly absorb the fluorescence, which decreases blue fluorescence intensity for BFP and CFP donors that can be erroneously interpreted as reduced donor fluorescence quantum yield caused by FRET [18]. If sensitized emission or FSPIM is the only available method for quantifying FRET, then it is very important to restrict measurements to chlorophyll free areas within the cells. 

Antibodies interact noncovalently with their target epitope, and the strength of this interaction is characterized by the kinetics of association and dissociation of the antibody. Antibody-antigen interactions are in principle reversible, and appropriate conditions must therefore be selected for a given antibody to bind with reproducible stoichiometry to its target antigen. Linkage of the antibody to an appropriate fluorochrome will mean that the number of antibody molecules bound will be reflected by the fluorescence intensity/cell. 

When the deoxynucleotide is associated with a fluorochrome, the cells can be observed under a fluorescence microscope, whereby apoptotic cells present an intense fluorescence and, in advanced stages, nuclear fragmentation can be visualized. For a quantitative analysis, either a hemocytometer in an optical microscope or a flow cytometer can be used (Tinto et al., 2002). Peroxidase-marked deoxynucleotides can be quantified by chromo-genic tests that use the enzyme substrate. Indirect methods use antigens linked to the nucleotide and recognized by labeled antibodies. However,... 

Two types of T4 lysozyme bindings to cell walls are possible (1) nonspecific attachment and (2) chemical binding associated with the hydrolysis reaction. Events indicated by the fluorescence intensity dropping to background level are associated with T4 lysozyme diffusing away from the cell wall. When the T4 lysozyme attached to the cell wall, many enzymatic reaction turnovers likely occurred in experiments, we have observed that the cell wall typically shrinks and eventually disappears from the imaging field of view [12]. 

Fig. 1. Coupling of the TAT-peptide to the distal end of PEG-chains on liposomes increases the cellular association with OVCAR-3 cells. OVCAR-3 cells are incubated with various concentrations rhodamine-PE labelled control (open circle) or TAT-liposomes (closed circle) for 1 h at 4°C, washed and analyzed by flow cytometry. Each data point represents the mean fluorescence intensity of 5,000 cells (mean SD 3). Error bars are within plot symbols when not visible. (Reproduced from (12) with permission from Elsevier Science)...

Taxonomic probes, including antibodies and nucleic acid or peptide nucleic acid (PNA) probes selectively stain particular target cells by association with antigens or DNA/RNA. The corresponding approaches are designated as immunofluorescence (IF) and fluorescence in situ hybridisation (FISH), respectively. The major limitation of these labelling procedures is the low fluorescence intensity, which results in poorly labelled cells that escape detection. 

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