Flow Cytometers

Flow cytometer cell counts are much more precise and more accurate than hemocytometer counts. Hemocytometer cell counts are subject both to distributional (13) and sampling (14—16) errors. The distribution of cells across the surface of a hemocytometer is sensitive to the technique used to charge the hemocytometer, and nonuniform cell distribution causes counting errors. In contrast, flow cytometer counts are free of distributional errors. Statistically, count precision improves as the square root of the number of cells counted increases. Flow cytometer counts usually involve 100 times as many cells per sample as hemocytometer counts. Therefore, flow cytometry sampling imprecision is one-tenth that of hemocytometry. 

Okada et al. examined the effects of TBT on cellular content of glutathione (GSH) in rat thymocites using a flow cytometer and 5-chloromethylfluorescein diacetate, a fluorescent probe for monitoring the change in the cellular content of GSH. TBT at nanomolar concentrations reduced the cellular content of GSH. There is an important implication on the TBT-induced depletion of cellular GSH since GSH has an important role in protecting the cells against oxidative stress and chemical and metal intoxications. TBT-induced decrease in cellular content of GSH in thymocytes may increase the vulnerability of the immune system. ° ... 

A flow cytometer equipped with forward-scattering and side-scattering detectors and a sorting option that can distinguish cell size-and-shape, sorting specified cells of 1 to 3pM length into small volumes of culture broth in individual plate wells. (This instrument is used for step 2 and in another mode may contribute to step 1.)... 

Figure 5.2 Synchronized, strobe-illuminated photograph showing the flow cytometer s liquid stream breaking into droplets that separate just as the stream enters a charge collar. ...

The flow cytometer, fitted with both forward and side scatter detectors, generates a 2D plot indicating the distribution of light intensity, forward scatter (FS) versus side scatter (SS), and showing the physical profile of the particle responsible for the scatter. Figure 5.3 is such a plot for a mixture of five rod-shaped bacteria from our laboratory. The different strains appear in partly separated clusters (indicated by square boxes and dot color) along the side-scatter axis in the lower part of the plot. 

Materials required As described in the principles, a researcher needs access to a flow cytometer plus fluorescent dyes generally, these are sold as intact emits, though the fluids used in the injection wells can differ - but they should not unduly alter the current between the electrodes lest confounding effects occur. 

Just as a Coulter Counter or a flow cytometer can automate or at least increase efficiency in counting heterospecific pollen transfer, use of artificial germination media still is an effective method of screening for pollen allelopathy. The fundamental approach recommended has not changed much in the last decade, i.e. sequentially dilute extracts from non-macerated... 

S. Gaward, L. Schild, and P. Renaud, Micromachined impedance spectroscopy flow cytometer for cell analysis and particle sizing. Lab on a Chip 1, 76-82 (2001). 

The digital data from the flow cytometer are collected in list mode files that contain a record of all of the fluorescent events associated with each individual cell in a sample. For any individual cell, the number of events collected is dependent on the number of fluorochromes utilized in the analysis that bind to the cell, which can be amplified by increasing the variety/number of lasers utilized to excite the fluorochrome. These raw data can then be analyzed on-line or off-line by software provided by the manufacturer of... 

FIGURE 7.8 Production of reactive oxygen intermediates by activated macrophages. Cells isolated from the livers of control (CTL) or toxicant (TOX)-treated mice were incubated with phorbol myristate acetate for 15 minutes at 37°C, followed by the indicator dye, DCFH-DA. After 15 minutes, the cells were analyzed for green fluorescence by flow cytometry on a Coulter Cytomics FC500 flow cytometer (Beckman Coulter). For each analysis, at least 10,000 events were collected and analyzed using CXP software. 

In addition to flow cytometers, cytometric instrumentation has evolved to include Laser Scanning Cytometry (LSC). The LSC is a cross between a flow cytometer and an imaging cytometer. Data are equivalent to the flow cytometer but is slide-based. Endpoints include light scatter and fluorescence, but the instrument can also record position of individual cells on the slide so that cells of interest can be relocated and re-evaluated or photographed. LSC is valuable when cell morphology is important. 

FIGURE 7.1 Representative diagram of the mechanics of a flow cytometer. For description,... 

Endou, H., Koseki, C., Kimura, K., Yokokura, Y., Fukida, S. and Sakai, F. (1982). Use of a flow cytometer for the separation of isolated kidney cells. In Biochemistry of Kidney Functions (Morel, F., ed.). Elsevier Biomedical Press, Amsterdam, pp. 69-78. 

Fig. 6 (a) Schematic illustration of a flow cytometer used in a suspension array. The sample microspheres are hydrodynamically focused in a fluidic system and read-out by two laser beams. Laser 1 excites the encoding dyes and the fluorescence is detected at two wavelengths. Laser 2 is used to quantify the analyte, (b) Scheme of randomly ordered bead array concept. Beads are pooled and adsorbed into the etched wells of an optical fiber, (c) Scheme of randomly-ordered sedimentation array. A set of encoded microspheres is added to the analyte solution. Subsequent to binding of the analyte, microparticles sediment and assemble at the transparent bottom of a sample tube generating a randomly ordered array. This array is evaluated by microscope optics and a CCD-camera. Reproduced with permission from Refs. [85] and [101]. Copyright 1999, 2008 American Chemical Society... 

Figure 6 is a schematic representation of a DNA histogram. The ability of the flow cytometer to rapidly count several thousand nuclei contributes to the sensitivity of this technique for DNA analysis. However, problems due to sample quality, staining, and instrumental artifacts should be recognized and minimized to insure accurate interpretation of data (B2). 

The difficulties of intensity-based flow cytometry are illustrated by the present difficulties of cell-by-cell measurements of intracellular calcium. This can be accomplished using the calcium probe Indo-l,(34 38) but requires an ultraviolet (UV) laser source which is not routinely available in flow cytometry (Indo-1 is an emission wavelength ratiometric probe). Flow cytometers routinely have argon ion laser sources with outputs of 488 or 514 nm. Measurement of intracellular ions other than Ca2+ is nearly impossible. (The SNAFL and SNARF probes should allow pH measurement from the wavelength-ratiometric data.(15))... 

Monoparametric DNA Analysis Fixed cells were stained with PI 25 pg/niL, RNase 5 pg/mL, Nonidet P 40 0.125 pg/mL. Cells were kept at RT for 60 in the dark and were analyzed by BD FACSCalibur flow cytometer. Aggregates were gating out by DDM papameters and DNA content analysis was performed in >10,000-gated cells. DNA histograms were analyzed using ModFit LT . 

BrdU Incorporation and DNA content Fixed cells were washed and resuspended with HC1 3 N for 20 . After washing with sodium tetraborate 0.1 M, cells were incubated with anti-BrdU antibody. After washing with PBS, cells were incubated with FITC goat anti-mouse IgG. After incubation, cells were washed and counterstained with PI 2 pg/mL and incubated overnight, and analyzed by BD FACSCalibur flow cytometer [20],... 

Caspase-3 Activation Analysis fixed cells were washed and incubated overnight with rabbit anti-active caspase-3 monoclonal antibody followed by FITC goat anti-rabbit antibody. Then cells were washed and mixed with PI and analyzed by BD FACSCalibur flow cytometer. For WB, cells are lysed with SDS and proteins were analyzed with caspase 3 Asp-175, p53 and yH2AX Ser-139. 

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