Tissue Cell Intracellular Immunofluorescent Staining kit 組織細胞內螢光染色試劑

組織細胞內螢光染色試劑

Tissue Cell Intracellular Immunofluorescent Staining kit

(Cat. No. FC024)

Introduction:

A modification of the basic immunofluorescent staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular antigens at the single-cell level. In this protocol, cells are first activated in vitro , stained for surface antigens as in the surface antigen protocol, then fixed with paraformaldehyde to stabilize the cell membrane and permeabilized with the detergent saponin to allow anti-cytokine antibodies to stain intracellularly. In vitro stimulation of cells is usually required for detection of cytokines by flow cytometry since cytokine levels are typically too low in resting cells. Stimulation of cells with the appropriate reagent will depend on the cell type and the experimental conditions. For example, to stimulate T cells to produce IFN-γ, TNF-α, IL-2, and IL-4, a combination of PMA (a phorbol ester / PKC activator) and Ionomycin (a calcium ionophore) or anti-CD3 antibodies can be used. To induce IL-6, IL-10 or TNF-α production by monocytes, stimulation of PBMC's with lipopolysaccharide (LPS) can be used.

In contrast to detection of secreted cytokines by ELISA, for detection of intracellular cytokines, it is necessary to block secretion of cytokines with protein transport inhibitors, such as Monensin or Brefeldin A . It is advised that the investigators evaluate the use and efficacy of different protein transport inhibitors in their specific assay system.

Flow cytometry requires the preparation of suspension of single cells. While it is comparatively easy with blood cells or cultured cells, the dispersion of intact cells from solid tissues is virtually impossible because cell junctions are very difficult to disrupt without damage to the cells membrane. We introduce this techniques that base on buffer with citric acid treatment that is applied either to fresh tissue samples or to archival material embedded in paraffin.

When using whole blood cell or spleen tissue cell suspensions for flow cytometric analysis, it is recommended to remove red blood cells. The kit is intended for lysing erythrocytes in single cell suspensions of hematopoietic tissues such as spleen and human peripheral blood. The buffer lysis red cells with minimal effect on lymphocytes which following direct immuno- fluorescence staining monoclonal antibodies prior to flow cytometric analysis. The cells may be stored for a month before analysis.

Contents of the kit: