Staining intracellular antigens like cytokines can be difficult because antibody-based probes cannot pass easily through the plasma membrane into the interior of the cell. In order to accomplish this, cells should first be fixed in suspension and then permeabilized before adding the fluorophore. This allows probes to access intracellular structures while leaving the morphological scatter characteristics of the cells intact. Triton is one example of a permeabilization reagent (Figure 18). There are also many commercial kits available today that provide the reagents to carry out these crucial steps, for example, Leucoperm™.
Figure. 18. 0.1% Triton used in conjunction with phalloidin, which recognizes and binds to filamentous actin. A, before treatment of cells; B, after fixation and permeabilization. Notice how distinctive the positive dataset becomes. FL, fluorescence.
Two-parameter or Bivariate Histograms
Chapter 4: Sample Preparation and Protocols
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