Flow Cytometry Sample Preparation Buffers and Reagents

Fixatives are necessary for saving samples to be used later or for looking at intracellular or intranuclear targets. Ready-to-use fixation kits are optimized for flow cytometry applications. Benefits of using these kits include the following:

• Methods used to stain cells take into consideration the location of the target proteins
• The fixation and permeabilization methods keep the morphological light-scattering characteristics of the cell intact
• Reagents in the kits help reduce background staining

Choosing the appropriate activating reagent will depend on (1) cell type, (2) expression level and kinetics of the protein of interest, and (3) experimental conditions.

Learn more about eBioscience Flow Cytometry Staining Buffer, Invitrogen FIX & PERM™ Cell Permeabilization Kit (RUO and GPR), Intracellular Fixation and Permeabilization Buffer Set (RUO), and the eBioscience Foxp3/ Transcription Buffer Set (RUO).

Activated cells often express higher levels of transcription factors, cytokines, chemokines, and other mediators detected by flow cytometry. Staining intracellular proteins for flow cytometry analysis often requires a fixation step to crosslink proteins and stabilize the cell membrane, as well as permeabilization to allow the antibodies access to the intracellular antigens. The Invitrogen eBioscience intracellular fixation and permeabilization buffers are designed for optimal detection of cytoplasmic and secreted proteins residing in organelles and vesicles. Benefits include:

• Compatible with a wide variety of markers and intracellular proteins
• Mild fixation and permeabilization for more accurate morphology

Learn more about eBioscience Flow Cytometry Staining Buffer and the Invitrogen FIX & PERM™ Cell Permeabilization Kit.

Figure 2. Staining of mouse CCL3 (MIP-1α) in macrophages. BALB/c thioglycolate-elicited peritoneal macrophages were unstimulated (left) or stimulated for 5 hours with LPS (Cat. No. 00-4976) (right) in the presence of Invitrogen eBioscience Protein Transport Inhibitor Cocktail (Cat. No. 00-4980). Cells were intracellularly stained with Anti–Mouse CD11b PerCP-Cyanine5.5 (Cat. No. 45-0112) and Anti–Mouse CCL3 (MIP-1α) PE (Cat. No. 12-7532) using the Invitrogen eBioscience Intracellular Fixation & Permeabilization Buffer Set (Cat. No. 88-8824) and protocol. Total viable cells, as determined by the Invitrogen eBioscience Fixable Viability Dye eFluor 450 (Cat. No. 65-0863), were used for analysis.

Flow cytometry permits the detection of transcription factors within discrete immune cell subsets among a heterogeneous population and provides a sensitive approach to analyzing an immune response. Refer to the following section on intracellular staining buffers prior to any transcription factor staining analysis. Benefits include:

• Compatibility with all fluorophores including polymer dyes such as Brilliant Violets and Super Bright dyes.
• Lower signal-to-noise ratio, helping with low frequency signal.
• FoxP3 staining buffer has been tested on many antibodies targeting transcription factors and optimized for nuclear protein staining.

Learn more about Foxp3 buffer, and see the data

Figure 3. Staining of mouse E4BP4 (NFIL3) in stimulated NK cells. Mouse splenocytes unstimulated (left) or stimulated overnight with Invitrogen eBioscience Mouse IL-15/IL-15R Complex Carrier- Free Recombinant Protein (Cat. No. 34-8152 [right]) were stained with Anti–Mouse NK1.1 PE-Cyanine7 (Cat. No. 25-5941) and Anti–Mouse E4BP4 (NFIL3) PE (Cat. No. 12-5927). Intracellular staining for E4BP4 was performed using the Invitrogen eBioscience Foxp3/Transcription Factor Staining Buffer Set (Cat. No. 00-5523) and protocol. Cells in the lymphocyte gate were analyzed.
 

*IVD—In Vitro Diagnostics Use
**GPR—General Purpose Reagent.