Graphic art showing a sample preparation

Flow cytometry buffers for sample preparation

Sample preparation is an important step for obtaining statistical value from flow cytometry experiments. Sample preparation reagents for flow cytometry include cell surface staining, intracellular and transcription factor staining buffer sets, cell lysis assays, blocking reagents, and magnetic cell isolation beads. Benefits of using these buffers include the following:

  • Retains inherent biological characteristics
  • Reduce background staining
  • Quicker and easier preparation with optimized kits

Read the table below to select the buffer for your experiment.

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Flow cytometry buffer selection

  Cal-Lyse Solutions High-Yield Lysis Solutions
Use
  • Premixed lysis solution formulated to lyse erythrocytes following monoclonal antibody staining of whole blood or bone marrow


Learn more about cell preservation and lysis buffers
  • Premixed, fixative-free erythrocyte-lysis solution that can be used to help eliminate red cells from whole blood for flow cytometric analysis with minimal loss of rare blood cell populations
Learn more about cell preservation and lysis buffers
Contents
  • Whole blood lysis buffer
  • Formaldehyde fixative
  • Fixative-free whole blood lysis buffer
Size 25 mL 100 mL 500 mL
Cat. No. GAS010 GAS010S100 HYL250
Regulatory status For In Vitro Diagnostic Use. For In Vitro Diagnostic Use.
When to use

Intended Use Statement:

Intended to be used as an aid in the enumeration of leukocytes from various human biological sources including blood and bone marrow that have been stained with monoclonal antibodies for analysis using flow cytometric methods.

Intended Use Statement:

Intended to be used to eliminate red cells from whole blood for flow cytometric analysis with minimal loss of rare blood cell populations.

  eBioscience Flow Cytometry Staining Buffer
Use
  • For antibody and cell dilution steps, as well as wash steps required for surface staining and flow cytometric analysis
  • Use with any cell type (human, mouse, etc.)
  • Volume can be scaled for use in both test tubes and plates
Learn more about the Flow Cytometry Staining Buffer
Contents Buffered saline solution:
  • Fetal bovine serum
  • Sodium azide (0.09%) as a preservative
Size 200 mL
Cat. No. 00-4222-57
  eBioscience Intracellular Fixation & Permeabilization Buffer Set FIX & PERM Cell Fixation & Cell Permeabilization Kit
Use
  • Stain both cytoplasmic proteins as well as proteins in the secretory pathway such as chemokines and cytokines
  • Not recommended for intranuclear staining
  • Use with any cell type (human, mouse, etc.)
  • Volume can be scaled for use in both test tubes and plates


Learn more about the IC Fix & Perm buffer set
  • Use when staining intracellular markers, such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, and immunoglobulins
  • Mild fixation and permeabilization of cells that leaves their morphological scatter characteristics intact
  • Use with any cell type (human, mouse, etc.)
  • Volume can be scaled for use in both test tubes and plates
Learn more about the FIX & PERM Kit
Contents
  • IC Fixation Buffer
  • Permeabilization Buffer (10X)
  • Fixation Medium (Medium A)
  • Permeabilization Medium (Medium B)
Size 1 kit 50 tests
Cat. No. 88-8824-00 GAS003
Regulatory status RUO – For Research Use Only. Not for Use in Diagnostic Procedures. GPR – General Purpose Reagent.
When to use For use in research only (i.e. research on any sample type). For use in general laboratory application, that is used to collect, prepare, and examine specimens from the human body for diagnostic purposes.
  eBioscience Foxp3 / Transcription Factor Staining Buffer Set
Use
  • Formulated and optimized for staining with antibodies to transcription factors and nuclear proteins, such as Foxp3 and Ki-67
  • Also applicable to simultaneous nuclear and cytoplasmic (cytokines and transcription factors) staining
  • Use with any cell type (human, mouse, etc.)
  • Volume can be scaled for use in both test tubes and plates
Learn more about the Foxp3 / Transcription Factor Staining Buffer Set
Contents
  • Fixation/Permeabilization Concentrate
  • Fixation/Permeabilization Diluent
  • Permeabilization Buffer (10X)
Size 1 kit
Cat. No. 00-5523-00
  eBioscience Super Bright Staining Buffer
Use
  • Use when more than one Super Bright dye–conjugated antibody is present in an experiment. This buffer helps prevent non-specific polymer interactions, which can result in data appearing under-compensated
  • Use with any cell type (human, mouse, etc.)
  • Volume can be scaled for use in both test tubes and plates
Learn about Super Bright Antibody Conjugates
Contents
  • Super Bright Staining Buffer
Size 100 tests
Cat. No. SB-4400-42
  Cal-Lyse Solutions High-Yield Lysis Solutions
Use
  • Premixed lysis solution formulated to lyse erythrocytes following monoclonal antibody staining of whole blood or bone marrow


Learn more about cell preservation and lysis buffers
  • Premixed, fixative-free erythrocyte-lysis solution that can be used to help eliminate red cells from whole blood for flow cytometric analysis with minimal loss of rare blood cell populations
Learn more about cell preservation and lysis buffers
Contents
  • Whole blood lysis buffer
  • Formaldehyde fixative
  • Fixative-free whole blood lysis buffer
Size 25 mL 100 mL 500 mL
Cat. No. GAS010 GAS010S100 HYL250
Regulatory status For In Vitro Diagnostic Use. For In Vitro Diagnostic Use.
When to use

Intended Use Statement:

Intended to be used as an aid in the enumeration of leukocytes from various human biological sources including blood and bone marrow that have been stained with monoclonal antibodies for analysis using flow cytometric methods.

Intended Use Statement:

Intended to be used to eliminate red cells from whole blood for flow cytometric analysis with minimal loss of rare blood cell populations.

  eBioscience Flow Cytometry Staining Buffer
Use
  • For antibody and cell dilution steps, as well as wash steps required for surface staining and flow cytometric analysis
  • Use with any cell type (human, mouse, etc.)
  • Volume can be scaled for use in both test tubes and plates
Learn more about the Flow Cytometry Staining Buffer
Contents Buffered saline solution:
  • Fetal bovine serum
  • Sodium azide (0.09%) as a preservative
Size 200 mL
Cat. No. 00-4222-57
  eBioscience Intracellular Fixation & Permeabilization Buffer Set FIX & PERM Cell Fixation & Cell Permeabilization Kit
Use
  • Stain both cytoplasmic proteins as well as proteins in the secretory pathway such as chemokines and cytokines
  • Not recommended for intranuclear staining
  • Use with any cell type (human, mouse, etc.)
  • Volume can be scaled for use in both test tubes and plates


Learn more about the IC Fix & Perm buffer set
  • Use when staining intracellular markers, such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, and immunoglobulins
  • Mild fixation and permeabilization of cells that leaves their morphological scatter characteristics intact
  • Use with any cell type (human, mouse, etc.)
  • Volume can be scaled for use in both test tubes and plates
Learn more about the FIX & PERM Kit
Contents
  • IC Fixation Buffer
  • Permeabilization Buffer (10X)
  • Fixation Medium (Medium A)
  • Permeabilization Medium (Medium B)
Size 1 kit 50 tests
Cat. No. 88-8824-00 GAS003
Regulatory status RUO – For Research Use Only. Not for Use in Diagnostic Procedures. GPR – General Purpose Reagent.
When to use For use in research only (i.e. research on any sample type). For use in general laboratory application, that is used to collect, prepare, and examine specimens from the human body for diagnostic purposes.
  eBioscience Foxp3 / Transcription Factor Staining Buffer Set
Use
  • Formulated and optimized for staining with antibodies to transcription factors and nuclear proteins, such as Foxp3 and Ki-67
  • Also applicable to simultaneous nuclear and cytoplasmic (cytokines and transcription factors) staining
  • Use with any cell type (human, mouse, etc.)
  • Volume can be scaled for use in both test tubes and plates
Learn more about the Foxp3 / Transcription Factor Staining Buffer Set
Contents
  • Fixation/Permeabilization Concentrate
  • Fixation/Permeabilization Diluent
  • Permeabilization Buffer (10X)
Size 1 kit
Cat. No. 00-5523-00
  eBioscience Super Bright Staining Buffer
Use
  • Use when more than one Super Bright dye–conjugated antibody is present in an experiment. This buffer helps prevent non-specific polymer interactions, which can result in data appearing under-compensated
  • Use with any cell type (human, mouse, etc.)
  • Volume can be scaled for use in both test tubes and plates
Learn about Super Bright Antibody Conjugates
Contents
  • Super Bright Staining Buffer
Size 100 tests
Cat. No. SB-4400-42


Cell isolation and expansion reagents

  Dynabeads for cell isolation MagniSort Cell Separation Technology
Use
  • Cell isolation
  • Cell activation and expansion
  • Clinical and non-clinical research
  • Cell isolation, including cell enrichment and depletion
Technology Dynabeads activation and expansion technology mimics in vivo T cell activation.

Available for human and mouse basic research as well as clinical research settings.

Learn more
Starting with T cell–specific cocktails, use the MagniSort platform to obtain enriched T cells simpler, faster and with significant cost-savings compared to traditional column-based separation methods.

Learn more


Flow cytometry buffers for intracellular and cytoplasmic staining

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).

Graphic art showing a sample preparation workflow for flow cytometry

Figure 1. Cytoplasmic and intracellular staining procedure. The location of the target proteins within the cell is an important consideration in the selection of the appropriate buffer and staining protocol. For cytokine or phosphorylated proteins, timing of stimulants and treatment with protein transport inhibitors are required steps before staining.


Cytoplasmic and cytokine staining reagent kits

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:

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

Two dot plots, with the left being unstimulated and right being stimulated with a shift of cells to the top right corner

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.


Nuclear staining reagent kits

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

Two dot plots, with the left being unstimulated and right being stimulated with a shift of cells to the top right corner

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.
 

5 Steps to Intracellular Flow Cytometry

Discover products and resources that are available to help optimize your intracellular flow cytometry experiments.
Best practices for performing intracellular flow cytometry

Resources

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Flow Cytometry Panel Builder—Design your flow cytometry panel with this online tool for a simplified, customizable experience to fit your needs.

5 Steps Resources

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Flow Cytometry Protocols

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Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.

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