Staining cells for internal antigens techniques have been devised for permeabilizing cells so that specific antibody can diffuse in and out and access internal antigens. Briefly, cells are fixed with formaldehyde to preserve their morphology, and saponin, a plant glycoside and mild nonionic detergent, is used to generate pores of approximately 8nm in the membrane which allow molecules of up to 200 kDa to pass through. Because of slow diffusion in and out of the cell, longer staining and washing times are required, cell and antibody preparations must be of high quality, and antibody must be at optimal titre to reduce nonspecific background. Other treatments can be used. For staining nucleic acid, fixation and permeabilization with alcohol and acetic acid reduces degradation of RNA and DNA. Cells may also be stained with antibody to a cell surface antigen for multicolour analysis. IntraCell is intended for fixation and permeabilization of cells in suspension for flow cytometry analysis. Immunological detection studies of intracellular antigens on structures such as cytoplasmic and/or nuclear enzymes, requires the permeabilization of the cell membrane in order to allow interaction of the antibody with its intended target.. In order to allow recognizable cellular integrity after permeabilization a fixation step involving cross-linking or denaturation is required. This is usually accomplished by aldehyde or alcohol fixation followed by detergent permeabilization of the cell membrane. Labeling of cell surface antigens with antibodies prior to the fixation step is possible thus allowing simultaneous surface antigens phenotyping with internal antigen expression in multiparameter fluorescent cytometric analysis. IntraCell is developed for use with all flow cytometers and reagents remains intact the cell surface marker expression and the cell properties of FSC and SSC.
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