Cell sorting by flow

Oltz, E.M., Pollack, S., Delohery, T, Smith, M.J., Ojika, M., Lee, S., Rustin, K and Nakanishi, K. (1989) Distribution of tunichrome and vanadium in sea squirt blood cells sorted by flow cytometry. Experientia, 45,186-190. 

Metaphase chromosomes can be sorted by flow cytometry in the same way that cells can be sorted (see 10.7.5). Usually separation is based on the DNA content of ethidium bromide or propidium iodide stained chromosomes but Hoechst 33258 (which preferentially stains AT rich segments) and chromomycin A3 (which stains CG rich regions) can also be used (Davies et al., 1981). 

Fig. 10.13. Distribution of cells separated by flow microfluorometry. CHO cells were pulse labelled for 15 min with [3H]thymidine (1 / Ci/ml) and stained with ethidium bromide. They were then submitted to flow microfluorometry and cell sorting on the basis of cellular DNA content. Cells from the indicated portions (sort 1, 2 and 3) were then subjected to autoradiography and shown to contain respectively 4%, 93% and 19% of the cells labelled. (Reproduced from Kraemer et al., 1973, with kind permission of the authors and publisher.)...

Testing can be performed on genomic DNA isolated from the entire ceU population of the specimen. Alternatively, the sample can be sorted by flow cytometry or immunorosetting to assess engraftment of specific ceil lineages. For example, engraftment of the T-cell versus non-T-cell components can be assessed after flow cytometry sorting based on expression of the CD3 surface molecule, a T-ceU marker. 

Figure 46-6. Flow of membrane proteins from the endoplasmic reticulum (ER) to the cell surface. Horizontal arrows denote steps that have been proposed to be signal independent and thus represent bulkflow. The open vertical arrows in the boxes denote retention of proteins that are resident in the membranes of the organelle indicated. The open vertical arrows outside the boxes indicate signal-mediated transport to lysosomes and secretory storage granules. (Reproduced, with permission, from Pfeffer SR, Rothman JE Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem 1987 56 829.)...

J. Porter, C. Edwards, J. A. W. Morgan, and R. W. Pickup, Rapid, automated separation of specific bacteria from lake water and sewage by flow cytometry and cell sorting, Appl. Environ. Microbiol. 59 3327 (1993). 

The chemical composition of particles can be just as varied as their shape. Commercial particles can consist of polymers or copolymers, inorganic constructs, metals and semiconductors, superparamagnetic composites, biodegradable constructs, and synthetic dendrimers and dendrons. Often, both the composition of a particle and its shape govern its suitability for a particular purpose. For instance, composite particles containing superparamagnetic iron oxide typically are used for small-scale affinity separations, especially for cell separations followed by flow cytometry analysis or fluorescence-activated cell sorting (FACS). Core-shell semiconductor particles, by... 

Analytical flow cytometry offers a rapid and facile means of monitoring cellular receptor content. For example, multiparameter flow cytometry techniques were used to monitor expression of GABAa receptor subunits during neurogenesis in embryonic rat brain (Marie et al., 2001). The content of the cell surface p75 neurotrophin receptor was measured in a heterogeneous population of mouse dorsal root sensory neurons, from which high and low p75 subsets were subsequently isolated by cell sorting (Barrett et al., 1998). 

Fig. 9.5. Efficiency and speed of sorting are affected by the flow rate of cells. At high flow rates, more desired cells are lost, but the speed of collecting these desired cells increases until the loss of efficiency becomes greater than the increase in speed. Highspeed sorting, with more drops per second, increases the efficiency and decreases the time required to obtain the desired number of cells. The model from which these graphs were generated was derived by Robert Hoffman for these data, a three-drop sort envelope was used, 1% of the cells were sorted, and the electronic dead time was set at 6 ps. If one drop is sorted with each sort decision (instead of three), the theoretical efficiency of the sorting improves considerably (as does the rate of collecting the sorted cells).

Fig. 6.3. Cutoff fluorescence selection for screening. Instrumentation, labeling, and biological noise introduce spreading into a fluorescence measurement, such that the fluorescence probability distributions for wild-type and mutant cells overlap. The logarithm of single-cell fluorescence as measured by flow cytometry is generally well-approximated by a symmetrical Gaussian curve. A cutoff fluorescence value is selected for screening, with all cells above that value sorted out. The enrichment factor forthe mutants is the ratio of (dotted + striped areas)/(striped area), and the probability of retention of a given mutant clone at a single pass is the (striped + dotted area)/(all area under mutant curve).

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