Flow Cytometry Protocols

Direct and indirect staining, staining of intracellular antigens, permeabilization and cell preparation protocols

The flow cytometry protocols below provide detailed procedures for the treatment and staining of cells prior to using a flow cytometer.

Protocols are available for:

• Direct staining of cells applicable where the fluorophore is directly linked to the primary antibody
• Indirect staining of cells applicable when using unconjugated or biotin-conjugated monoclonal and polyclonal antibodies
• Intracellular staining methods for intracellular antigens and cytokines
• DNA staining for cell cycle analysis
• Cell preparation

Direct Staining Flow Cytometry Protocols

This is one of the simplest and most common staining methods, where live or fixed cells are incubated with directly labeled antibodies against cell surface antigens

1. Direct immunofluorescence staining surface epitopes of cells and blood
2. Direct immunofluorescence staining of immunoglobulin light chains on B lymphocytes in whole blood

Indirect Staining Flow Cytometry Protocols

If there is not a directly labeled antibody available, or you wish to amplify your signal you can do indirect staining. This is where you stain a cell with a primary antibody against the antigen of interest and visualize using a labeled secondary antibody which recognizes the primary.

1. Indirect immunofluorescence staining of surface epitopes of cells and blood

Intracellular Staining Flow Cytometry Protocols

In order to detect antigen not present on the cell surface, cells have to be fixed and permeabilized to disrupt the cell membrane and allow entry of the antibody. Antigens can be then directly or indirectly labeled. Various methods are optimal depending on the antigen and antibody used.

1. Direct staining of intracellular antigens: Leucoperm™ Accessory Reagent method
2. Direct immunofluorescence staining of intracellular cytokines in blood
3. Direct immunofluorescence staining of intracellular antigens: Methanol plus Leucoperm™ Accessory Reagent method
4. Direct immunofluorescence staining of intracellular antigens: Digitonin method
5. Direct immunofluorescence of intracellular antigens: Paraformaldehyde / Saponin method

Cell Cycle Staining Flow Cytometry Protocols

Measuring DNA content for cell cycle analysis requires fixation and permeabilization of the nuclear membrane. We have protocols for staining using DNA binding dyes with and without antibody staining and a protocol for BrdU staining.

1. Immunofluorescence staining of cells in combination with PI staining of cells for cell cycle analysis
2. Propidium iodide staining of cells for cell cycle analysis
3. BrdU staining of cells for cell cycle analysis

Cell Preparation Flow Cytometry Protocols

Below are protocols for harvesting cells from various sources to obtain healthy cells, essential for optimal staining and analysis.

1. Preparation of human peripheral blood mononuclear cells
2. Preparation of cells for flow cytometry
3. Preparation of peritoneal macrophages, bone marrow, thymus and spleen cells

Product Specific Flow Cytometry Protocols

Specific protocols to use with our antibodies.

1. BrdU staining of cells with Mouse Anti-BrdU Antibody (clone Bu20a), by flow cytometry
2. Measuring cell proliferation using CytoTrack™ cell proliferation assay kit

Cell Activation Protocols

General activation protocols using pharmacological reagents and antibodies. Ideal to determine immune competence, marker upregulation, cytokine release and proliferation by flow cytometry.

1. Unprimed T cell activation - pharmacologic method
2. Unprimed T cell activation - antibody stimulation method
3. Primed T cell activation - antigen presenting cell co-culture

Flow Cytometry Products

• View all flow cytometry antibodies
• View all flow cytometry isotype controls

Flow Cytometry Resources

• Controls in Flow Cytometry
• Flow Cytometry guide
• Fluorochromes
• Intracellular Flow Cytometry
• Tips for the design of multi-color flow panels
• Technical support