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# Goal/Experiment:
**Activation and Intracellular Staining of Whole Blood: For the Detection of Intracellular Cytokines and Other Intracellular Targets**

## BioLegend, Inc.

### Abstract
This is part of BioLegend's, "Intracellular Flow Cytometry Staining Protocol: For the Detection of Intracellular Cytokines and Other Intracellular Targets."

### Guidelines

#### Application Notes

1. **Activated Cell Preparation:**
   - Activated cell populations can be prepared from in vivo-stimulated tissues or vitro-stimulated cultures (e.g., antigen-specific activation or mitogen-induced).
   - For cytokine and chemokine detection, include a protein transport inhibitor such as brefeldin A (BioLegend Cat. No. 420601) or monensin (BioLegend Cat. No. 420701) in the last 4-6 hours of cell culture activation.
   - The cells can be suspended and distributed to 12 x 75 mm plastic tubes or microwell plates for immunofluorescent staining.

2. **Optimization of Stimulation Conditions:**
   - Different cytokines/chemokines have different production peaks. Stimulation conditions for each stimulant should be optimized.

3. **Surface Marker Antibodies:**
   - Some antibodies recognizing native cell surface markers may not bind to fixed/denatured antigens.
   - It is recommended that staining of cell surface antigens be done with live, unfixed cells PRIOR to fixation/permeabilization and staining of intracellular targets. 

#### Note
To confirm specific anti-cytokine staining, a blocking experiment is recommended:
- Cells fixed/permeabilized then preincubated with an excess amount of unlabeled anti-cytokine antibody and/or the recombinant cytokine of interest is preincubated with fluorophore-conjugated anti-cytokine antibody before its addition to the cells.

### Related Information

1. Assenmacher, M., et al. 1994. Eur. J. Immunol. 24:1097.
2. Elson, L.H., et al. 1995. J. Immunol. 1995. 154:4294.
3. Jung T, et al. 1993. J. Immunol. Methods 159:197.
4. Prussin C., et al. 1995. J. Immunol. Methods 188:117.
5. Vikingsson A., et al. 1994. J. Immunol. Methods 173:219.

### Reagent List

1. **Cell Staining Buffer** (BioLegend Cat. No. 420201)
2. **Monensin** (BioLegend Cat. No. 420701)
3. **RBC Lysis Buffer** (BioLegend Cat. No. 420301)
4. **Brefeldin A** (BioLegend Cat. No. 420601)
5. **Fixation Buffer** (BioLegend Cat. No. 420801)
6. **Intracellular Staining Perm Wash Buffer** (BioLegend Cat. No. 421002)
7. **Cyto-Last™ Buffer** (BioLegend Cat. No. 422501)

### Protocol

#### Activation

**Step 1.**
- Dilute heparinized whole blood 1:1 with sterile appropriate tissue culture medium.

**Step 2.**
- Perform in vitro cellular stimulation by either antigen or mitogen.
- If staining intracellular cytokines or chemokines (e.g., IFN-γ or IL-4), add a protein transport inhibitor such as brefeldin A (BioLegend Cat. No. 420601) or monensin (BioLegend Cat. No. 420701).

**Step 3.**
- Aliquot 200 µl of the whole blood cell suspension into 12 x 75 mm plastic tubes.
- Incubate for 4-6 hours in 5% CO2 at 37°C.

**Step 4.**
- Add 2 ml of 1X Red Blood Cell Lysis Buffer (BioLegend Cat. No. 420301) and incubate for 5-10 minutes at room temperature.

**Step 5.**
- Centrifuge at 350 x g for 5 minutes and discard the supernatant.

**Step 6.**
- Wash cells 1X with Cell Staining Buffer.

**Step 7.**
- If staining intracellular antigens (e.g., IFN-γ or IL-4), first perform cell surface antigen staining as described in BioLegend’s [Cell Surface Immunofluorescence Staining Protocol](https://biolegend.com) then fix cells in 0.5 ml/tube Fixation Buffer (BioLegend Cat. No. 420801) in the dark for 20 minutes at room temperature.

**Step 8.**
- Centrifuge at 350 x g for 5 minutes and discard the supernatant.

**Step 9.**
- To put the experiment "on hold" for future staining and analysis:
  - Wash cells 1x with Cell Staining Buffer (BioLegend Cat. No. 420201).
  - Resuspend cells in Cell Staining Buffer and store cells at 4°C (short term) or in 90% FCS/10% DMSO for storage at -80°C (long term, for fixed cells without surface antigen staining).

  _Note: Alternatively, cells can be kept in Cyto-Last™ Buffer (BioLegend Cat. No. 422501) for the storage of cytokine-producing cells for up to two weeks._

#### Permeabilization

**Step 10.**
- Dilute 10X Intracellular Staining Perm Wash Buffer (BioLegend Cat. No. 421002) to 1X in DI water.

**Step 11.**
- Resuspend fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5-10 minutes. (1/3)

**Step 12.**
- Resuspend fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5-10 minutes. (2/3)

**Step 13.**
- Resuspend fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5-10 minutes. (3/3)

#### Intracellular Staining

**Step 14.**
- Resuspend fixed/permeabilized cells in residual Intracellular Staining Perm Wash Buffer and add a predetermined optimum concentration of fluorophore-conjugated antibody of interest (e.g., PE anti-IFN-γ) or an appropriate negative control for 20 minutes in the dark at room temperature.

**Step 15.**
- Wash with 2 ml of Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5 minutes. (1/2)

**Step 16.**
- Wash with 2 ml of Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5 minutes. (2/2)

**Step 17.**
- If primary intracellular antibody is biotinylated, perform fluorophore-conjugated Streptavidin incubations and subsequent washes in Intracellular Staining Perm Wash Buffer.

**Step 18.**
- Resuspend fixed and intracellularly labeled cells in 0.5 ml Cell Staining Buffer and analyze with appropriate controls.

#### Flow Cytometric Analysis

**Step 19.**
- Set PMT voltage and compensation using cell surface staining controls.
- Set quadrant markers based on blocking controls, isotype controls, or unstained cells.

  _Note: For proper flow cytometric analysis, cells stained by this method should be inspected by light microscopy and/or flow light scatter patterns to confirm that they are well dispersed._

  _Note: Bivariate dot plots or probability contour plots can be generated upon data analysis to display the frequencies of and patterns by which individual cells coexpress certain levels of cell surface antigen and intracellular cytokine proteins._

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