How do you do intracellular staining?

Flow cytometry can be used to analyze various intracellular molecules including phosphorylated signaling proteins and cytokines. To stain intracellular molecules, the cells need to be fixed in suspension and then permeabilized before the detection antibody is added.

What is FACS protocol?

Fluorescent activated cell sorting (FACS) is a specialized type of flow cytometry used for sorting and analyzing a heterogeneous mixture of cells into different subpopulations based on the specific light scattering and fluorescent characteristics (from the specific labels) of each cell.

What is intracellular cytokine staining ICS?

Intracellular cytokine staining (ICS) is a method of cytokine analysis that provides information on the type of cytokines that are produced upon (antigen-specific) stimulation of T cells and B cells.

What is the difference between surface and intracellular staining in flow cytometry?

One advantage of exploiting surface antigens is that live cells can be subjected to cell sorting paradigms such as FACS. In contrast, intracellular antigen staining requires fixation and permeabilization steps to mediate the epitope-antibody interaction, precluding downstream applications that require viable cells.

How does FC block work?

Fc Blocking. Flow cytometry utilizes fluorescently labeled antibodies to bind and identify specific cellular subsets. The specificity of the binding relies on the unique variable regions of each antibody clone.

Why do we wash cells after staining?

washing helps removal of unbound antibodies, so that the second fluorescent antibody can efficiently label primary antibody.

What is Annexin V staining?

Annexin V staining is a common method for detecting apoptotic cells. Thermo Fisher Scientific offers high-quality fluorescent annexin V conjugates as standalone reagents and in a variety of kits for use in flow cytometry and for imaging suspension cells.

Is FC block necessary?

To stain T-cells, blocking Fc receptors is not essential.

What is tetramer staining?

A tetramer assay (also known as a tetramer stain) is a procedure that uses tetrameric proteins to detect and quantify T cells that are specific for a given antigen within a blood sample.

Can you fix after intracellular staining?

You can fix the surface stains and then do the intracellular staining step the next day if you are worried about losing ICS signal. Hello, Fix the cells and covered with aluminium foil and placed at 4°C, you can run on flow cytometer within the next couple of days (briefly vortex before running).