Preparation of cell suspensions from solid tissues and cell cultures
Cell suspensions can readily be prepared from monolayers of cultured cells, e.g. human umbilical vein endothelial cells (HUVECs) by the dissociation procedures used when passaging cells (see below). They can also be prepared from solid mammalian tissues by disrupting cell-cell junctions and/or the interactions between cells and the
intercellular matrix. Depending on the tissue concerned this may be achieved by enzyme treatment and/or mechanical means. Mechanical disaggregation alone, is really only suitable for those tissues in which the cells are loosely bound, e.g. bone marrow, spleen and lymph nodes. It is much less effective for tissues in which cells are tightly bound and enzymes are normally used instead to disrupt the bonds which bind cells to the extracellular matrix and to each other. Proteases, e.g. trypsin, collagenase, elastase and more recently dispase (a metallo- neutral-protease with a mild action), and enzymes which hydrolyse the extra-cellular matrix, e.g. hyaluronidase and lysosyme, are commonly used to separate cells. Chelating agents (EDTA or citrate) are often included in order to remove the Ca 2+ and Mg 2+ ions that are essential for matrix stability and cell-matrix interactions but will not liberate cells alone. They can be used in conjunction with trypsin but not collagenase because activity of the latter is Ca 2+ -dependent. DNase is often included to prevent cell-cell aggregation which might otherwise be induced by the DNA released from damaged cells.
The aim is to obtain a good yield of cells, whilst simultaneously preserving the plasma membrane and intra-cellular components. If proteases are used, the possibility that they will cleave, and thereby alter the antigenicity of, cell surface glycoproteins must always be borne in mind. The viability of the dispersed cells obtained from solid tissue is generally higher following enzymic than mechanical dissociation but the former can lead to selected losses of certain cell populations. If cell suspensions are to be used for flow cytometry, they should be filtered through nylon mesh, or single cells recovered by density gradient centrifugation (e.g. on Ficoll-Hypaque), to avoid blocking the flow cell. As there are few comparative studies of the merits of different dissociation procedure, it is advisable to monitor the products both by cytometry and microscopy to confirm that the cells are representative of the original tissue. A factor which is of particular importance when sampling heterogeneous tumours. A mechanical tissue disaggregation system, designed specifically for preparing samples for flow cytometry is available commercially (Medimachine System; BD Biosciences). It would be particularly useful where solid tissue samples are frequently analysed as it facilitates standardisation and removes variability in processing technique.
Fixed samples Fixation techniques and potential artefacts
Fixation is clearly not appropriate for functional studies but has been used extensively in conjunction with immunolabelling for phenotypic analysis. There are many potential advantages to fixing cells at some stage during the preparation procedure. Cells in samples that are fixed soon after collection and prior to labelling, are theoretically not subject to inadvertent post-collection changes (e.g. the up-regulation of adhesion molecule expression on peripheral blood neutrophils) which might otherwise occur in vitro . Moreover, they can often be processed later, at a perhaps more convenient time. However, fixation before labelling also has some drawbacks. Most important is the likelihood that antigenicity will be altered (usually diminished), leading to decreased antibody binding or even false-negative results. In addition, the 'signal to noise' ratio can be decreased because the non-specific binding of antibodies is increased.
When contemplating the use of fixatives it is as well to understand their mode of action. All commonly used fixatives cause chemical changes in nucleic acids and proteins which result in conformational changes that can alter, or abolish, antigenicity. In general, antigenicity is lost progressively with increasing concentration of fixative and increasing time of fixation, whilst ultrastructural integrity is increasingly preserved. Consequently, the conditions chosen for fixation are always a compromise between preserving ultrastructure and losing antigenicity. There are two main types of fixatives: protein precipitants/coagulants and cross-linking agents. Alcohols and acetone are precipitating or coagulating fixatives. Traditional cross-linking fixatives include the aldehydes such as formaldehyde but new bi-functional molecules initially developed for protein biochemistry studies are increasingly being used. Dialdehyde fixatives such as glutaraldehyde, which is widely used in electron microscopy, are successful because they form cross-links almost as rapidly as they form adducts. However, they cause a high background autofluorescence which makes them unsuitable for many cytometric applications. The newer carbodiimide cross-linking reagents act by linking carboxyl to amino groups through amide (peptide) bond formation. Although not yet widely used they have the advantage of producing only low background fluorescence.