FACS analyser

2 Choice of antibodies, reagents, and general maintenance of the FACS analyser 

Commercially available lysis solution (e.g. Ortho Lysis, Ortho Diagnostics), supplied anhydrous, is best used to remove RBC because it is quality controlled by the manufacturer. Measmed aliquots which reconstitute to 100 ml are made in dean water (Elga, Reverse Osmosis water purifier) and used within two weeks. 

Although some manufacturers recommend saline for the sheath buffer of 

Target ceil Antigenic determinant (positivity)-fiuorochrome 

FACS analysers, dean water is preferable because it does not cause blockages in the andllary tubing due to deposition of salts induding calcium. 

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Fluorescence-Activated Cell Sorter (FACS) Analyses... 

These sorts of analyses are limited only by the ability of the chosen mass spectrometer to detect the test-compound under infusion-buffer conditions. In many cases, there will not be a problem even at low nanomolar compound concentrations, but in others MS will be hard-pressed to detect compounds even at micromolar concentrations. The online FAC-MS system will always be challenged by the inherent incompatibility of routine assay buffers with MS, but there are opportunities to reconfigure either the ion source or the buffer composition for... 

Mass spectrometry enables the type of direct analyses described, but it does have its limitations. Online operation forces detection at infusion concentrations, in salty buffer and under complex mixture conditions. General ion suppression results from the buffer and mixture components, and mixture complexity can tax the resolution of even the best mass spectrometers. Increasing compound concentration is not the answer, as this leads to problems of solubility and increased compound consumption. We have found that the online method can work successfully for up to 100 compounds per analysis, but the false negative rate becomes appreciable [21]. As an alternative for ligand discovery purposes, we have developed a FAC-LC/MS system in which FAC effluent is sampled and analyzed by LC/MS [19]. This system offers the ability to concentrate mixture components and introduces another dimension to the data in order to tolerate more complex mixtures (Fig. 6.9). Using this system, we have screened approximately 1000 modified trisaccharide acceptor analogs targeting immobilized N-... 

Whatever the application, MS-based analyses of FAC effluent will always be faced with the need to support a wide range of buffer components, ranging from variable ionic strength, surfactant levels to required cofactors. In select situations such as indicator analyses, online methods may be appropriate but it is clear that the insertion of an intermediate LC step offers sigmficantly improved performance. This changes the nature of the data analysis, from the detection of sigmoidal breakthrough curves to peak detection and differential analysis across multiple fractions. 

A computer program was developed (Dean and Jett, 1974) to resolve the flow microfluorometric distribution into Gl, S and G2 + M subpopulations and enable cell cycle analyses to be performed rapidly and accurately. This has been constantly improved and is incorporated on the software of modern FACS machines (e.g. the Becton Dickinson FACScan) (see 11.4.3 and Fig. 11.3). Some... 

Cell sorting takes place at a rate of 300,000 cells/min and for this reason a FACS machine is more usually used as an analytical tool rather than in a preparative mode. In addition to its use in cell cycle analysis, it can be used (a) to analyse the distribution of lymphocytes carrying a series of different surface antigens (e.g. to determine the proportion of T4 lymphocytes in a blood sample) (b) to estimate the proportion of dead cells in a population (i.e. cells which stain with propidium iodide without prior fixation) or (c) to determine the proportion of transformed cells (i.e. cells bearing a particular surface antigen) in a culture or biopsy (Watson, 1987). 

Finally, the cell pellet is suspended in 5 00 pi FACS buffer and analysed for doxorubicin fluorescence using a FACS instrument (here FACS Vantage microflow cytometer, Beckton Dickinson) using the 488 nm line of an air cooled argon laser as the excitation source. Fluorescence from cell associated doxorubicin is detected using a 550 nm long pass emission filter. 

Different types of analyses for the detection of micronuclei originating from structural or numerical aberrations are used light microscopy, semiautomated imaging systems, and flow cytometry (FACS analysis) [66, 67], Fluorescent in situ hybridization (FISH) with specific probes (e.g., centromeric probes) can also be used to examine nondisjunctions. Depending on the methodology, specific features of the damage can be characterized and mechanisms of action addressed. 

C. Kanony and M. Mokrane, Reconstitution de la courbe des taux, analyse des fac-teurs d evolution et couverture factorielle, Cahiers de la Cai e Autonome de Refi-nancement 1 (June 1992). France (1989-90)—Spot ZC 1Y-25Y 2 93.7/6.1... 

Table 1 Immunophenotype of lymphocyte subsets and stem cells, visualized by RTC or PE readyconjugated mAbs and analysed by FACS...

The percentage of different WBC in peripheral blood is shown in Table 3. Since the lymphocyte component is approximately 20-40% of the nucleated cell count, FACS analysis of lymphocyte subsets is done with approximately 10000 cells yielding an appropriate number of lymphocytes to analyse. In contrast, since the number of CD34 cells is often less that 1% of WBC in blood. BM, and PBSC harvests, quantitation requires at least 20000 ceU to be analysed. 

Before examining blood, BM, or PBSC harvests with mAbs, the scatter properties of whole blood are used to standardize the analyser that has been assessed for the differential count by other methods (e.g. Coulter Counter ACT-8)). Computer software is provided by most FACS manufiicturers to assist with the calibration. This procedure enables the mononuclear cell (MNC) and lymphocyte gates to be identified, so that there is > 95% agreement between the two methods. 

Take 0.4 ml. Count and analyse for T cell subsets and CD34 cells by FACS (Protocol I). 

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