Best Practices for Intracellular Flow Cytometry

5 steps of 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.

Target determination ►

Find intracellular target antibody conjugates quickly and easily 

Buffer selection ►

Achieve the best possible results with highly referenced sample preparation reagents

Panel design ►

Pair your antibodies and fluorophores efficiently using best practices

Biology controls ►

Remove dead cells and unwanted artifacts during analysis

Experimental controls ►

Correct for overlapping fluorescence emission spectra


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.

  • Buffers for fixation and permeabilization have significant impact on the quality and accuracy of data
  • See the difference for yourself using our optimized staining buffers

Cytoplasmic staining

  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.

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)
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.

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)
Size 1 kit
Cat. No. 00-5523-00

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

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 assay LIVE/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.
Readout Dead 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 compatibility Cannot be used in combination with Invitrogen product line such as Indo-1 (blue), DAPI, or Hoechst 33342 Cannot be used in combination with Invitrogen product line such as Pacific Blue dye, CellTrace Violet stain, FxCycle Violet stain, BV421 or eFluor 450 Cannot be used in combination with Invitrogen product line such as Pacific Green dye, F2N12S apoptosis assay, AmCyan, or BV510 Cannot 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
Size 80 tests
Cat. No.

488 & 561 nm lasers

  LIVE/DEAD Fixable Green stain LIVE/DEAD Fixable Red stain
Basis of assay LIVE/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.
Readout Dead 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 compatibility Cannot be used in combination with Invitrogen product line such as FITC, Alexa Fluor 488, GFP, or CellTrace CSFE stain Cannot 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
Size 80 tests
Cat. No.

633/635 nm laser

  LIVE/DEAD Fixable Far Red stain LIVE/DEAD Fixable Near-IR stain
Basis of assay LIVE/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.
Readout Dead 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 compatibility Cannot be used in combination with Invitrogen product line such as APC, Alexa Fluor 647 dye, FxCycle Far Red and CellTrace Far Red stains Cannot 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
Size 80 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
Use Designed 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.
Reactivity Hamster, mouse, and rat antibodies and recognition of the kappa & lambda chains
Format One vial, dispense as a single drop
Laser compatibility Compatible with most standard lasers, UV to 633 nm
Size 25 tests
Cat. No. 01-2222-41

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.

  • Buffers for fixation and permeabilization have significant impact on the quality and accuracy of data
  • See the difference for yourself using our optimized staining buffers

Cytoplasmic staining

  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.

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)
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.

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)
Size 1 kit
Cat. No. 00-5523-00

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

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 assay LIVE/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.
Readout Dead 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 compatibility Cannot be used in combination with Invitrogen product line such as Indo-1 (blue), DAPI, or Hoechst 33342 Cannot be used in combination with Invitrogen product line such as Pacific Blue dye, CellTrace Violet stain, FxCycle Violet stain, BV421 or eFluor 450 Cannot be used in combination with Invitrogen product line such as Pacific Green dye, F2N12S apoptosis assay, AmCyan, or BV510 Cannot 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
Size 80 tests
Cat. No.

488 & 561 nm lasers

  LIVE/DEAD Fixable Green stain LIVE/DEAD Fixable Red stain
Basis of assay LIVE/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.
Readout Dead 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 compatibility Cannot be used in combination with Invitrogen product line such as FITC, Alexa Fluor 488, GFP, or CellTrace CSFE stain Cannot 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
Size 80 tests
Cat. No.

633/635 nm laser

  LIVE/DEAD Fixable Far Red stain LIVE/DEAD Fixable Near-IR stain
Basis of assay LIVE/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.
Readout Dead 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 compatibility Cannot be used in combination with Invitrogen product line such as APC, Alexa Fluor 647 dye, FxCycle Far Red and CellTrace Far Red stains Cannot 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
Size 80 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
Use Designed 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.
Reactivity Hamster, mouse, and rat antibodies and recognition of the kappa & lambda chains
Format One vial, dispense as a single drop
Laser compatibility Compatible with most standard lasers, UV to 633 nm
Size 25 tests
Cat. No. 01-2222-41