Cell labeling/separation

A fluorescence-activated cell sorter (FACS) is a flow cytometry instrument used to separate and identify cells in a heterogeneous population. Cell mixtures to be sorted are first bound to fluorescent dyes such as fluorescein or phycoerythrin. The labeled cells are then pumped through the instrument and are excited by a laser beam. Cells that fluoresce are detected, and an electrostatic charge is applied. The charged cells are separated using voltage deflection. 

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.)...

Apart from assays of proteolytic activity and immunochemical methods (7.1 and 7.5), protein labeling is exploited in other areas of analysis. Noteworthy among them are protein determination in complex mixtures or even directly in cells, protein separation (affinity chromatography and electrophoresis) and protein detection following various separation techniques. For this purpose different labels are applied. 

The uptake kinetics of C-labelled triadimenol, shown in figure 6 were examined in both the susceptible and triadimenol-resistant strains of the yeast Saccharomycopsis lipolytica. To minimize the effect of unspecific absorption on the cell surface the incubated cells were separated by suction filtration and washed twice with unlabelled triadimenol. The passive uptake curve shows the typical characteristics of saturation by diffusion, equilibrium being reached after about 20 minutes. There are no real differences between the susceptible and resistant isolates, and no indication of transport mutation or induction of efflux transport can be derived from these data. 

Rembaum, A. and Dreyer, W.J. (1980) Immunomicrosphere reagents for cell labeling and separation, Science, 208, 364-368... 

Cellular labeling/cell separation Cell labeling with MNPs is a method for in vivo cell separation, as the labeled cells can be detected by magnetic resonance imaging (MRI) [187, 188]. 

In magnetic cell sorting (MACS), cells labeled with primary antibody are incubated with a ferrous conjugated second antibody. Labeled cells are separated by decanting the cell suspension in a magnetic field. Pizzonia et al. [101] and Bacskai and co-authors [102] introduced MACS for immunoselection of murine distal tubule cells. Baer et al. [103] used MACS for im-munodissection of human proximal and distal tubule... 

A big additional advantage of flow-through processes in comparison with stationary processes consists in the possibility of implementation of process chains (Fig. 7). In this way, complete microanalytical systems can be designed [13]. They can include sample pretreatment like cell separation, cell lysis, or chemical separation. Reverse transcription processes can be directly ctamected on-chip with the DNA amplification by thermocycling (RT-PCR). The analytical chain can also include post-amplification steps like fluorescence labeling, separation by capillary electrophoresis, or hybridization on DNA chips. 

Fig. 3. Effect of 3-methoxybenzamide on the appearance of the T-cell receptor during the development of mouse thymic lobes in organ culture. Thymuses were removed from 14-day mouse embryos and were maintained in vitro in the presence and absence of 3-methoxybenzamide (18). After 12-14 days, thymocytes were harvested and labelled with monoclonal antibody F-23.1 which recognizes the products of a V B gene family present on about 25% of peripheral T cells (22). The percentage of cells which were surface-labelled with the antibody was determined by fluorescence microscopy. The clear bars indicate the percentage of cells labelled with F-23.1 in cultures maintained continuously in the indicated concentrations of 3-methoxybenzamide. Filled bars show the corresponding percentage in control cultures run in parallel. The standard errors of the indicated number of separate experiments (n) is given in each case. Each experimental and control result was obtained using a minimum of 5 thymic lobes.

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