Fixation and Permeabilization Solution
- Brand BD Cytofix/Cytoperm™
Intracellular staining (flow cytometry) (Routinely Tested)
- Regulatory Status RUO
Regulatory Status Legend
BD Cytofix/Cytoperm™ solution is supplied as a 1X solution and can be used for the simultaneous fixation and permeabilization of cells prior to intracellular cytokine staining.
Suggested Companion Products
Preparation and Storage
Store undiluted at 4°C.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
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.
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.
May cause respiratory irritation.
Wear protective clothing / eye protection.
Wear protective gloves.
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.
If skin irritation or rash occurs: Get medical advice/attention.