For the stimulation of cells, various in vitro methods have been reported for stimulating cells to produce cytokines. Polyclonal activators have been particularly useful for inducing cytokine-producing cells. These activators include the following: concanavalin A, lipopolysaccharide, phorbol esters plus calcium ionophore or ionomycin, phytohaemaglutinin, staphylococcus enterotoxin B, and monoclonal antibodies directed against subunits of the TCR/CD3 complex (with or without antibodies directed against costimulatory receptors, such as CD28).
1. Fix and Permeabilize Cells
a. Thoroughly resuspend cells in 100 µL of BD Cytofix/Cytoperm solution per well for microwell plates (or 250 µL for tubes) and incubate for 20 min. at 4°C. Note: Cell aggregation can be avoided by vortexing prior to the addition of the BD Cytofix/Cytoperm™ solution.
b. Wash cells two times in a buffer that contains a cell permeabilizing agent such as saponin (BD Perm/Wash™ buffer, Cat. 554723, which
can be used as the wash buffer and as the antibody diluent).
2. Stain for Intracellular Cytokines
a. Thoroughly resuspend fixed/permeabilized cells in 50 µL of a saponin-containing buffer (e.g., BD Perm/Wash™ buffer) containing a pre- determined optimal concentration of a fluorochrome-conjugated anti-cytokine antibody or appropriate negative control. Incubate at 4°C for
30 minutes in the dark. Note: Because saponin-mediated cell permeabilization is a reversible process, it is important to keep the cells in the
presence of saponin during intracellular cytokine staining.
b. Wash cells 2 times with saponin-containing buffer (e.g, BD Perm/Wash™ buffer) and resuspend in staining buffer prior to flow cytometric analysis.
Note: Both the BD Cytofix/Cytoperm™ (Cat. No. 554722) and BD Perm/Wash buffer (Cat. No. 554723) are included in the Fixation/Permeabilization Solution Kit (Cat. No. 554714) as well as the Fixation/Permeabilization Solution Kit with BD GolgiStop
(containing monensin); Cat. No. 554715) and Fixation/Permeabilization Solution Kit with GolgiPlug (containing brefeldin A); Cat.
Danger: BD Cytofix/Cytoperm™ Buffer (Fixation and Permeabilization Solution) contains 4.2% formaldehyde (w/w).
Harmful if inhaled.
Causes skin irritation.
Causes serious eye damage.
May cause an allergic skin reaction.
Suspected of causing genetic defects.
May cause cancer. Route of exposure: Inhalative.
Harmful to aquatic life.
Obtain special instructions before use. Do not handle until all safety precautions have been read and understood.
Wear protective clothing / eye protection/ gloves.
Wash thoroughly after handling.
Do not breathe mist/vapours/spray.
IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
If skin irritation or rash occurs: Get medical advice/attention.
IF ON SKIN: Wash with plenty of water. Immediately call a POISON CENTER/doctor.
Wash contaminated clothing before reuse. Contaminated work clothing must not be allowed out of the workplace.
Avoid release to the environment..
Store locked up.
Dispose of contents/container to an appropriate treatment and disposal facility in accordance with applicable laws and regulations, and product characteristics at time of disposal.