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5 Steps to 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.
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.
Cytoplasmic staining
| eBioscience Intracellular Fixation & Permeabilization Buffer Set | FIX & PERM Cell Fixation & Cell Permeabilization Kit | |
|---|---|---|
| Use |
Learn more about the IC Fix & Perm buffer set |
|
| Contents |
|
|
| 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 |
|
| Contents |
|
| Size | 1 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.
- 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 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.
- 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 |
| 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.
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.
Cytoplasmic staining
| eBioscience Intracellular Fixation & Permeabilization Buffer Set | FIX & PERM Cell Fixation & Cell Permeabilization Kit | |
|---|---|---|
| Use |
Learn more about the IC Fix & Perm buffer set |
|
| Contents |
|
|
| 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 |
|
| Contents |
|
| Size | 1 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.
- 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 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.
- 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 |
| Compatible with most standard lasers, UV to 633 nm | |
| Size | 25 tests |
| Cat. No. | 01-2222-41 |
.
Getting started with our free panel design service
We have a team of technical support scientists available to help you. Our team provides this panel design service for those who want help with building their panel every step of the way.
Get expert help and pricing to put together your panels
Contact our technical sales specialists for help choosing antibodies, fluorophores, and reagents for success on the flow cytometer you’ll be using.
Resources
Fluorophore and reagent selection guide for flow cytometry
Download Flow Cytometry Protocols Handbook
Spectral Flow Cytometry Fundamentals
Invitrogen eBioscience Resources—Selection guides, Best Protocols, product performance and more.
Intracellular Staining for Flow Cytometry How-To Video—for detecting cytokines and intranuclear markers.
Flow Cytometry Learning Center—Access flow cytometry educational resources for better experiment planning and execution.
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
Support
Flow Cytometry Support Center—Find technical support recommendations for your flow cytometry workflows, including tips for experimental setup and in-depth troubleshooting help.
Flow Cytometry Panel Design Support—Work with one of our technical sales specialists to discuss your experimental needs and guide you through the process.