By Nicholas Catsimpoolas
Almost immediately, the necessity for tactics regarding separation, identity, and characterization of other sorts of cells is abundantly discovered between immu nologists, hematologists, mobile biologists, medical pathologists, and melanoma researchers. except cells displaying diversified features and levels of vary entiation are separated from each other, it will likely be enormously tricky to check the various molecular mechanisms taken with phone popularity, spe cialization, interactions, cytotoxicity, and transformation. medical diagno sis of diseased states and use of remoted cells for healing (e. g. , immu notherapy) or survival (e. g, transfusion) reasons are many of the urgent parts the place quick functional merits may be got by means of utilizing mobile separation thoughts. although, the improvement of such worthy equipment remains to be in its infancy. a few stable suggestions exist dependent both at the actual or organic homes of the cells, and those have produced a few important effects. nonetheless others are to be chanced on. for this reason, the aim of this open-ended treatise is to acquaint the reader with a few of the simple rules, instrumentation, and strategies almost immediately in perform at quite a few laboratories all over the world and to offer a few ordinary purposes of every strategy to specific organic difficulties.
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4. , 1951). Figure 7A demonstrates that mixtures of silica and PVP give very high background color. The final concentration of PVP in the mixture is responsible for the absorbance. Silica and MCS particles do not, however, interfere with the Lowry reaction to any large extent. Experiments with a protein standard are shown in Fig. 7B. , 1972). Higher concentrations of proteins can be determined with the biuret reaction (Dittebrandt, 1948). 5. Enzyme Assays Various cell organelles are usually identified by the presence of specific enzymes.
Experiment performed in collaboration with Dr. B. ,~ ~ ~ ~ :t CENTRIFUGATION IN MODIFIED COLLOIDAL SILICA GRADIENTS 45 Olsson and Pertoft (1967) resolved human bone marrow cells into a number of different subpopulations, and Ostberg (personal communication) has recently obtained a similar fractionation of bone marrow cells from mice. 2. Separation of Cells from Solid Tissues and Tissue Cultures Cells from solid tissues must be dispersed before they can be separated on a gradient. This is usually performed with mechanical or enzymatic means.
LAURENT o---Q I I / I I I \ \ \ \ ~ \l I /o_-o--d I I I I I I I I ? 0"'<). 10 FIGURE 13. The density distribution of cells from mouse spleen. The percentage of total cells at each density level is indicated (e_ e) as well as the percentage of cells reactive with 1311_ labeled rabbit antibodies against mouse immunoglubulin (B-cells) (0_0). 07 glml) to a final cell concentration of 1 x 1000/mi. Density gradient centrifugation was performed at 10,000 rpm for IS min in a Beckman rotor 65 at 20°C. 5-ml fractions, the number of cells were counted, and the radioactivity was measured by liquid scintillation.