Best Practices for Intracellular Flow Cytometry

Best practices for intracellular flow cytometry

The ability to stain and detect intracellular molecules opens the door to identify distinct cell subsets as well as further characterize cell populations. Our resources, tools and products help you save time and effort and increase your efficiency when designing your flow cytometry experiments.

Save time and energy by using our streamlined approach to find the antibody that you need quickly and easily.

Find your Antibody

When performing intracellular staining for flow cytometry the selection of buffers used for fixation and permeabilization has a significant impact on the quality and accuracy of data. We provide optimized solutions for staining cytosolic, nuclear and secreted proteins for flow cytometry.

Learn more: Intracellular Staining Quick Guide

Learn more: Intracellular Staining Buffer Selection Guide

Cytoplasmic staining

 eBioscience Intracellular Fixation & Permeabilization Buffer SetFIX & 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.

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.
Learn more about the FIX & PERM Kit
Contents
  • IC Fixation Buffer
  • Permeabilization Buffer (10X)
  • Fixation Medium (Medium A)
  • Permeabilization Medium (Medium B)
Size1 kit50 tests
Cat. No.88-8824-00GAS003
Regulatory statusRUO–For Research Use Only. Not for Use in Diagnostic Procedures.GPR–General Purpose Reagent.
When to useFor 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.

Nuclear staining

 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.
Learn more about the Foxp3 / Transcription Factor Staining Buffer Set
Contents
  • Fixation/Permeabilization Concentrate
  • Fixation/Permeabilization Diluent
  • Permeabilization Buffer (10X)
Size1 kit
Cat. No.00-5523-00

Web Tool: Flow Cytometry Panel Builder

  • Ideal for anyone designing a flow cytometry panel
  • Simplified, customizable panel design experience
  • Efficient antibody selection built on spectra visualization

Article: Flow Cytometry Panel Design–The Basics

  • Ideal read for anyone new to designing flow cytometry panels
  • Learn the key points to consider when designing a flow cytometry panel
  • Key literature references about panel design cited

Web reference: Optimized Flow Cytometry Multiplex Panels

  • Useful for all experience levels
  • Learn about optimized multicolor immunofluorescence panels (OMIPs)
  • Search existing published flow cytometry panels
  • Leverage a published panel to save time

Webinar: Basics of Multicolor Flow Cytometry Panel Design

  • Ideal for beginners, 1-hour run time
  • Dr. Holden T. Maecker of Stanford University discusses the caveats of good panel design including:
    • Examples and practical applications
    • Controls and standardization
    • Relevance of panel design to mass cytometry platforms

Save time and avoid inaccurate analysis of your cell population by removing dead cells and unwanted artifacts efficiently with the Invitrogen LIVE/DEAD Fixable Dead Cell Stains.

Biology controls
  • Fixation and permeabilization of cells results in cell death and often leads to unwanted artifacts
  • Save time and avoid inaccurate analysis of your cell population
  • Remove these dead cells and any unwanted artifacts efficiently

Find LIVE/DEAD kits for:UV & 405 nm lasers | 488 & 561 nm lasers | 633/635 laser

UV & 405 nm lasers

 LIVE/DEAD Fixable Blue
stain		LIVE/DEAD Fixable Violet
stain		LIVE/DEAD Fixable Aqua
stain		LIVE/DEAD Fixable Yellow
stain
Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
UV
405
405
405
Ex/Em (nm)
350/450
416/451
367/526
400/575
Bibliography
Citations
Multiplexable
Yes
Yes
Yes
Yes
Dye compatibilityCannot be used in combination with Invitrogen product line such as Indo-1 (blue), DAPI, or Hoechst 33342Cannot be used in combination with Invitrogen product line such as Pacific Blue dye, CellTrace Violet stain, FxCycle Violet stain, BV421 or eFluor 450Cannot be used in combination with Invitrogen product line such as Pacific Green dye, F2N12S apoptosis assay, AmCyan, or BV510Cannot be used in combination with Invitrogen product line such as Pacific Orange dye, Qdot 605, F2N12S apoptosis assay, or BV605
Fixable
Yes
Yes
Yes
Yes
Sample type
Live cells
Size80 tests
Cat. No.

488 & 561 nm lasers

 LIVE/DEAD Fixable Green stainLIVE/DEAD Fixable Red stain
Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
488
488
561
Ex/Em (nm)
495/520
595/615
Bibliography
Citations
Multiplexable
Yes
Yes
Dye compatibilityCannot be used in combination with Invitrogen product line such as FITC, Alexa Fluor 488, GFP, or CellTrace CSFE stainCannot be used in combination with Invitrogen product line such as R-PE, propidium iodide, SYTOX AADvanced stain, 7-AAD, PE-Alexa Fluor 610 and PE-Texas Red tandems
Fixable
Yes
Yes
Sample type
Live cells
Size80 tests
Cat. No.

633/635 nm laser

 LIVE/DEAD Fixable Far Red stainLIVE/DEAD Fixable Near-IR stain
Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
Laser (nm)
633/635
633/635
Ex/Em (nm)
650/665
750/775
Bibliography
Citations
Multiplexable
Yes
Yes
Dye compatibilityCannot be used in combination with Invitrogen product line such as APC, Alexa Fluor 647 dye, FxCycle Far Red and CellTrace Far Red stainsCannot be used in combination with Invitrogen product line such as APC-Alexa Fluor 750, APC-Cy7 or Vybrant DyeCycle Ruby stain
Fixable
Yes
Yes
Sample type
Live cells
Size80 tests
Cat. No.
L34973
L34975

Staining intracellular targets for flow cytometry requires the use of multiple antibodies that often have overlapping fluorescence emission spectra. The emission spillover must be compensated for correctly in order to ensure that the fluorescent signal being measure is due to the fluorochrome of interest and thus the cell population of interest.

Experimental controls
  • Save time setting your experimental antibody compensation controls correctly and easily
  • Supplied in a single vial—one drop is all you need to set an antibody compensation control

Learn more about eBeads microspheres for antibody compensation

     UltraComp eBeads
    UseDesigned for compensation of dyes in immunophenotyping experiments using fluorescently labeled antibodies, the beads can be stained with individual fluorochrome-conjugated antibodies for use as single-color compensation controls.
    ReactivityHamster, mouse, and rat antibodies and recognition of the kappa & lambda chains
    FormatOne vial, dispense as a single drop
    Laser compatibility Compatible with most standard lasers, UV to 633 nm
    Size25 tests
    Cat. No.01-2222-41
    Step 1 : Target determination

    Save time and energy by using our streamlined approach to find the antibody that you need quickly and easily.

    Find your Antibody

    Step 2 : Buffer selection

    When performing intracellular staining for flow cytometry the selection of buffers used for fixation and permeabilization has a significant impact on the quality and accuracy of data. We provide optimized solutions for staining cytosolic, nuclear and secreted proteins for flow cytometry.

    Learn more: Intracellular Staining Quick Guide

    Learn more: Intracellular Staining Buffer Selection Guide

    Cytoplasmic staining

     eBioscience Intracellular Fixation & Permeabilization Buffer SetFIX & 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.

    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.
    Learn more about the FIX & PERM Kit
    Contents
    • IC Fixation Buffer
    • Permeabilization Buffer (10X)
    • Fixation Medium (Medium A)
    • Permeabilization Medium (Medium B)
    Size1 kit50 tests
    Cat. No.88-8824-00GAS003
    Regulatory statusRUO–For Research Use Only. Not for Use in Diagnostic Procedures.GPR–General Purpose Reagent.
    When to useFor 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.

    Nuclear staining

     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.
    Learn more about the Foxp3 / Transcription Factor Staining Buffer Set
    Contents
    • Fixation/Permeabilization Concentrate
    • Fixation/Permeabilization Diluent
    • Permeabilization Buffer (10X)
    Size1 kit
    Cat. No.00-5523-00
    Step 3 : Panel Design

    Web Tool: Flow Cytometry Panel Builder

    • Ideal for anyone designing a flow cytometry panel
    • Simplified, customizable panel design experience
    • Efficient antibody selection built on spectra visualization

    Article: Flow Cytometry Panel Design–The Basics

    • Ideal read for anyone new to designing flow cytometry panels
    • Learn the key points to consider when designing a flow cytometry panel
    • Key literature references about panel design cited

    Web reference: Optimized Flow Cytometry Multiplex Panels

    • Useful for all experience levels
    • Learn about optimized multicolor immunofluorescence panels (OMIPs)
    • Search existing published flow cytometry panels
    • Leverage a published panel to save time

    Webinar: Basics of Multicolor Flow Cytometry Panel Design

    • Ideal for beginners, 1-hour run time
    • Dr. Holden T. Maecker of Stanford University discusses the caveats of good panel design including:
      • Examples and practical applications
      • Controls and standardization
      • Relevance of panel design to mass cytometry platforms
    Step 4 : Biology controls

    Save time and avoid inaccurate analysis of your cell population by removing dead cells and unwanted artifacts efficiently with the Invitrogen LIVE/DEAD Fixable Dead Cell Stains.

    Biology controls
    • Fixation and permeabilization of cells results in cell death and often leads to unwanted artifacts
    • Save time and avoid inaccurate analysis of your cell population
    • Remove these dead cells and any unwanted artifacts efficiently

    Find LIVE/DEAD kits for:UV & 405 nm lasers | 488 & 561 nm lasers | 633/635 laser

    UV & 405 nm lasers

     LIVE/DEAD Fixable Blue
    stain    		LIVE/DEAD Fixable Violet
    stain    		LIVE/DEAD Fixable Aqua
    stain    		LIVE/DEAD Fixable Yellow
    stain
    Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
    ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
    Laser (nm)
    UV
    405
    405
    405
    Ex/Em (nm)
    350/450
    416/451
    367/526
    400/575
    Bibliography
    Citations
    Multiplexable
    Yes
    Yes
    Yes
    Yes
    Dye compatibilityCannot be used in combination with Invitrogen product line such as Indo-1 (blue), DAPI, or Hoechst 33342Cannot be used in combination with Invitrogen product line such as Pacific Blue dye, CellTrace Violet stain, FxCycle Violet stain, BV421 or eFluor 450Cannot be used in combination with Invitrogen product line such as Pacific Green dye, F2N12S apoptosis assay, AmCyan, or BV510Cannot be used in combination with Invitrogen product line such as Pacific Orange dye, Qdot 605, F2N12S apoptosis assay, or BV605
    Fixable
    Yes
    Yes
    Yes
    Yes
    Sample type
    Live cells
    Size80 tests
    Cat. No.

    488 & 561 nm lasers

     LIVE/DEAD Fixable Green stainLIVE/DEAD Fixable Red stain
    Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
    ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
    Laser (nm)
    488
    488
    561
    Ex/Em (nm)
    495/520
    595/615
    Bibliography
    Citations
    Multiplexable
    Yes
    Yes
    Dye compatibilityCannot be used in combination with Invitrogen product line such as FITC, Alexa Fluor 488, GFP, or CellTrace CSFE stainCannot be used in combination with Invitrogen product line such as R-PE, propidium iodide, SYTOX AADvanced stain, 7-AAD, PE-Alexa Fluor 610 and PE-Texas Red tandems
    Fixable
    Yes
    Yes
    Sample type
    Live cells
    Size80 tests
    Cat. No.

    633/635 nm laser

     LIVE/DEAD Fixable Far Red stainLIVE/DEAD Fixable Near-IR stain
    Basis of assayLIVE/DEAD Fixable Dead Cell Stain kits are fixable viability dyes that distinguish live cells from dead cells based on cell membrane integrity and access to available amines. Cells can then be fixed for intracellular antigen detection without loss of original cell staining pattern. Allows easy exclusion of dead cells from flow cytometry readout.
    ReadoutDead cells are highly fluorescent and easy to distinguish from live cells, which are stained with minimal fluorescence
    Laser (nm)
    633/635
    633/635
    Ex/Em (nm)
    650/665
    750/775
    Bibliography
    Citations
    Multiplexable
    Yes
    Yes
    Dye compatibilityCannot be used in combination with Invitrogen product line such as APC, Alexa Fluor 647 dye, FxCycle Far Red and CellTrace Far Red stainsCannot be used in combination with Invitrogen product line such as APC-Alexa Fluor 750, APC-Cy7 or Vybrant DyeCycle Ruby stain
    Fixable
    Yes
    Yes
    Sample type
    Live cells
    Size80 tests
    Cat. No.
    L34973
    L34975
    Step 5 : Experimental controls

    Staining intracellular targets for flow cytometry requires the use of multiple antibodies that often have overlapping fluorescence emission spectra. The emission spillover must be compensated for correctly in order to ensure that the fluorescent signal being measure is due to the fluorochrome of interest and thus the cell population of interest.

    Experimental controls
    • Save time setting your experimental antibody compensation controls correctly and easily
    • Supplied in a single vial—one drop is all you need to set an antibody compensation control

    Learn more about eBeads microspheres for antibody compensation

       UltraComp eBeads
      UseDesigned for compensation of dyes in immunophenotyping experiments using fluorescently labeled antibodies, the beads can be stained with individual fluorochrome-conjugated antibodies for use as single-color compensation controls.
      ReactivityHamster, mouse, and rat antibodies and recognition of the kappa & lambda chains
      FormatOne vial, dispense as a single drop
      Laser compatibility Compatible with most standard lasers, UV to 633 nm
      Size25 tests
      Cat. No.01-2222-41

      .

      Cy™ is a trademark or registered trademark of GE Healthcare.

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

      Brilliant Ultra Violet™ and Brilliant Violet™ are trademarks or registered trademarks of Becton, Dickinson and Company or its affiliates, and are used under license.

      For Research Use Only. Not for use in diagnostic procedures.

      Hero banner dark gradient overlay