Intracellular Immunofluorescent Staining for Flow Cytometric Analysis (FACS Analysis)

• Intracellular Cytokine Staining Protocol
• Foxp3 Staining Protocol

Introduction

A modification of the basic immunofluorescent staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular antigens at the single-cell level. In this protocol, cells are first activated in vitro, stained for surface antigens as in the surface antigen protocol, then fixed with paraformaldehyde to stabilize the cell membrane and permeabilized with the detergent saponin to allow anti-cytokine antibodies to stain intracellularly. In vitro stimulation of cells is usually required for detection of cytokines by flow cytometry since cytokine levels are typically too low in resting cells. Stimulation of cells with the appropriate reagent will depend on the cell type and the experimental conditions. For example, to stimulate T cells to produce IFN-γ, TNF-α, IL-2, and IL-4, a combination of PMA (a phorbol ester / PKC activator) and Ionomycin (a calcium ionophore) or anti-CD3 antibodies can be used. To induce IL-6, IL-10 or TNF-α production by monocytes, stimulation of PBMC’s with lipopolysaccharide (LPS) can be used.

Note: Optimal stimulation period for induction of a given cytokine is variable and has to be determined. For example, the best time for detection of IL-6-producing cells by human LPS-activated monocytes is 6 hours, whereas IL-10 detection needs at least 24 hours stimulation.

In contrast to detection of secreted cytokines by ELISA, for detection of intracellular cytokines, it is necessary to block secretion of cytokines with protein transport inhibitors, such as Monensin or Brefeldin A, during the last few hours of the stimulation. It is advised that the investigators evaluate the use and efficacy of different protein transport inhibitors in their specific assay system.

Note: Generally, the non-specific background staining of fixed and permeabilized cells is higher than surface staining; therefore, extra protein such as BSA or FCS can be included in the staining buffer. The optimal concentration of the fluorochrome-conjugated anti-cytokine antibodies has to be determined experimentally. To confirm specificity of the staining, it is useful to block the directly-conjugated anti-cytokine antibodies with excess amounts of cognate ligand.

Buffers used for intracellular staining can have varying effects. eBioscience antibodies are optimized using eBioscience Foxp3 staining buffer set (cat. 00-5523) or eBioscience IC Fixation Buffer (cat. 00-8222). All antibodies to cytokines tested have stained appropriately using the Foxp3 buffers. Please contact Technical support for more information.

Granzyme B & IL-17 Data

Mouse/Human Granzyme B (GB11): Left: Human peripheral blood cells were stimulated for 2 days with IL-2. The cells were surface stained with FITC CD56 (MEM188) (Cat. No. 11-0569) and stained intracellularly with PE GB11. Quadrants demarcate boundary for isotype controls. Right: Mouse splenocytes were stimulated for 3 days with IL-2. Cells were surface stained with APC CD49d (DX5) (Cat. No. 17-5971) and subsequently stained intracellularly with PE GB11.			Human IL-17 (eBio64CAP17): Human PBMCs were stimulated with TPA/ionomycin in the presence of monensin for 5 hours. The cells were fixed and permeabilized and intracellularly stained with anti-human CD4, clone RPA-T4, (Cat. No. 17-0049) and Alexa Fluor® 488 anti-human IL-17 (Cat. No. 53-7178). Isotype control on the left and anti-IL-17 on the right. Cells in the lymphocytes gate were used for analysis.

Intracellular Staining Quick Guides

Intracellular Cytokine Staining Protocol

Precautionary Note: Prior to using antibodies, do a quick spin to recover the maximum volume from the vials. We monitor our conjugated antibodies to make sure they are void of aggregation. Some protocols include a step of airfuging the antibody prior to staining to further clear aggregates. For optimal performance of fluorochrome conjugated antibodies, store at 4°C in the dark. Do not freeze. When staining, please note that fixation and/or delayed analysis may yield poor signal-to-noise for certain antigens. For best results, stain fresh mouse cells and analyze soon after staining.

Materials

• Directly fluorochrome-labeled antibodies (see charts above) or Th1/Th2 Flow Panel
• Cells to be stained
• 12x75 mm round-bottom test tubes (Falcon Cat. No. 2008) or 96-well round-bottom microtiter plates

Buffers

• eBioscience IC Fixation Buffer (Cat. No. 00-8222)
• eBioscience Permeabilization Buffer (Cat. No. 00-8333)
• eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222)

Instruments

• Pipettes and pipettors
• Centrifuge
• Ice bucket or refrigerator
• Flow Cytometer

Experiment Duration

• Stimulation (variable depending on the cytokine of interest)
• 2-3 hours staining

Method

1. Prepare target cells of interest (see specific instructions).
2. Stain cell-surface antigen. The choice of the cell surface marker depends on the experimental question. For suggestions on best phenotypic markers for different cell types, please see the appropriate Mouse Phenotyping Markers Chart or Human Phenotyping Markers Chart.
3. After the last wash, fix the cells by adding 100µl of Fixation Solution, while vortexing the tube. Incubate in the dark at room temperature for 20 minutes.
4. Add 1ml of Permeabilization Buffer to each tube, centrifuge for 5 minutes and aspirate supernatant.
5. Repeat step 4.
6. Resuspend cells in 100µl of Permeabilization Buffer.
7. Dilute the optimal concentration of fluorochrome-labeled anti-cytokine mAb in 20µl Permeabilization Buffer and add to the appropriate tube. Mix by vortexing. A good range of antibody concentrations for this application is 0.5-0.06µg/10⁶ cells, 100µl staining volume. eBioscience fluorochrome-labeled anti-cytokine antibodies are also available in 20µl/test sizes. If using these reagents, add 20µl of pre-titrated antibody to the appropriate tube.
8. Incubate in the dark at room temperature for 20 minutes.
9. Add 1ml of Permeabilization Buffer, centrifuge for 5 minutes and aspirate supernatant.
10. Resuspend the cell pellet in 0.5ml of Flow Cytometry Staining Buffer.
11. Analyze samples on a flow cytometer.

Foxp3 Staining Protocol

The following protocol is for intracellular staining of Human Foxp3 (clone PCH101, cat. 4776). Please refer to specific clones for the corresponding protocol. We have also determined that this kit is suitable for most cytokine staining procedures.

It is critical to use the Foxp3 Staining Buffer Set (cat. 00-5523). The buffer set is included with all Foxp3 Staining Sets.

Prior to staining, dilute the Fixation/Permeabilization Concentrate (1 part) into the Fixation/Permeabilization Diluent (3 parts) to the desired volume of Fixation/Permeabilization working solution. This buffer should not be stored for more than 1 day. For example: For 12 samples, use 3 ml Fixation/Permeabilization Concentrate and 9 ml Fixation/Permeabilization Diluent.

1. Add 100 µl of prepared cells (1x10⁶) to each tube.
2. Stain surface molecules such as CD4, CD8, CD25, etc. following the Surface Staining Protocol (http://www.ebioscience.com/ebioscience/appls/FCS.htm).
3. Wash in cold Flow Cytometry Staining Buffer (or cold PBS).
4. Resuspend cell pellet with pulse vortex and add 1 ml of freshly prepared Fixation/Permeabilization working solution to each sample. Pulse vortex again.
5. Incubate at 4°C for 30 - 60 minutes in the dark.
6. Wash once by adding 2 ml 1X Permeabilization Buffer (made from 10X Permeabilization Buffer) followed by centrifugation and decanting of supernatant.
7. Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
8. [OPTIONAL] Block with 2% (2 µl) normal rat serum in 1X Permeabilization Buffer, in approximately 100 µl volume, at 4°C for 15 minutes.
9. Without washing after blocking step, add 20 µl fluorochrome conjugated anti-human Foxp3 antibody or isotype control in 1X Permeabilization Buffer and incubate at 4°C for at least 30 minutes in the dark. Please perform further titration for optimal staining in your own assay system.
10. Wash cells with 2 ml 1X Permeabilization Buffer. Centrifuge and decant supernatant.
11. Repeat step 10.
12. Resuspend in appropriate volume Flow Cytometry Staining Buffer and analyze on cytometer. Please note that due to the fixation and permeabilization procedure, the FSC/SSC distribution of the cell population will be different than live cells. Therefore the gate and voltages will need to be modified.