Protocol of Intracellular Staining Flow Cytometry

In order to detect intracellular antigen, cells have to be fixed and permeabilized to disrupt the cell membrane and allow entry of the detection antibody. Antigens can be then directly or indirectly labeled. Various methods are optimal depending on the antigen and antibody used. Please refer to the corresponding sections according to your experimental requirements.

• Protocol A: Protocol of Direct Immunofluorescence Staining of Intracellular Cytokines in Blood
• Protocol B: Protocol of Direct Immunofluorescence Staining of Intracellular Antigens: Digitonin Method
• Protocol C: Protocol of Direct Immunofluorescence of Intracellular Antigens: Paraformaldehyde / Saponin Method

These provided protocols are only guidelines and may need to be adjusted for particular applications. Adaptations may be necessary for specific protein targets within the cells. Refer to our Troubleshooting of Intracellular Staining Flow Cytometry. Please contact us for additional help if necessary.

Protocol A: Protocol of Direct Immunofluorescence Staining of Intracellular Cytokines in Blood

This is a simple and rapid method to the analysis of intracellular cytokines in whole blood. Directly conjugated antibodies is used in the assay. Besides, this method permits the analysis of small samples and avoids generating artefacts due to the separation of peripheral blood cells by density gradient centrifugation. All blood samples must be collected into heparin anticoagulant.

Reagents

• Regent A: Cell fixation agent
• Reagent B: Cell permeabilization agent
• PBS-BSA: PBS containing 1% BSA
• Red blood cell lysing buffer
• Cell culture medium
• Ionomycin
• Monensin
• PMA
• Optional: 0.5% (w/v) paraformaldehyde in PBS

Methods

1. Aliquot 500 μl of blood into as many tubes as required, including 2 extra control tubes, then add 500 μl of cell culture medium (without any additives) to each sample.
2. For the resting population, add monensin to a final concentration of 3 μM.
3. For the activated cells, add PMA to a final concentration of 10 ng/ml, ionomycin (2 μM) and monensin (3 μM).
4. For the experimental samples, add monensin (3 μM) and treat as required by the experiment.
5. Incubate for 3 hours at 37°C in a 5% CO₂ atmosphere.
6. If required, perform staining of cell surface antigens using appropriate directly conjugated monoclonal antibodies (mAbs) at 4°C. Avoid direct light.
7. Wash cells once with PBS-BSA. Discard supernatant.
8. Add 100 ul of Reagent A and incubate for 10 minutes at 2-8°C.
9. Add 2 ml pre-cold PBS-BSA and centrifuge for 5 minutes at 400 g at RT. Discard supernatant.
10. Add 100 ul Reagent B/1 x 10⁶ cells. Add the directly conjugated antibody at the vendor-recommended dilution and incubate for at least 30 minutes at 4°C. Avoid direct light.
11. Add 2 ml freshly prepared red blood cell lysing buffer to the blood suspension. Mix thoroughly.
12. Incubate for 10 minutes at RT.
13. Centrifuge at 400 g for 5 minutes. Discard the supernatant.
14. Wash once in PBS-BSA, and then resuspend in 200 μl PBS for immediate analysis or with 200 μl of 0.5% formaldehyde in PBS if required.
15. Acquire data by flow cytometry.
16. Data analysis.

Notes:

• Resting cells often need stimulation in vitro prior to the detection of intracellular cytokines.
• EDTA interferes with the cell stimulation process and therefore must be avoided.

Protocol B: Protocol of Direct Immunofluorescence Staining of Intracellular Antigens: Digitonin Method

This procedure causes a reduction in forward scatter (FSC) signal, so the flow cytometer set up may need to be adjusted to compensate.

Reagents

• PBS
• PBS-BSA: PBS containing 1% BSA
• 0.5% (w/v) paraformaldehyde in PBS
• 0.05% (v/v) Tween 20 in PBS
• 10 μg/ml digitonin in PBS

Methods

1. Prepare cells appropriately. Refer to Cell Preparation for Flow Cytometry and adjust the cell suspension to a concentration of 1 x 10⁷ cells/ml with cold PBS-BSA.
2. Aliquot 100 μl of the cell suspension into required number of tubes.
3. If required, perform staining of cell surface antigens using appropriate directly conjugated mAbs at 4°C. Avoid direct light.
4. Wash twice in pre-cold PBS-BSA by pelleting cells at 400 g and 4°C for 5 minutes. Discard supernatant.
5. Add 50 μl PBS-BSA to each tube, followed by 100 μl 0.5% paraformaldehyde. Incubate for 20 minutes at RT.
6. Wash twice with 3 ml 0.05% Tween 20. Pellet cells at 400 g and 4°C for 5 min. Discard supernatant.
7. Add 100 ul of cold 10 ug/ml digitonin and the directly conjugated antibody at the vendor-recommended dilution and incubate for at least 30 minutes at 4°C. Avoiding direct light.
8. Wash twice with 3 ml 0.05% Tween 20. After each wash step, pellet cells at 400 g and 4°C for 5 minutes.
9. Resuspend in 200 μl PBS.
10. Acquire data by Flow Cytometry.
11. Data analysis.

Protocol C: Protocol of Direct Immunofluorescence of Intracellular Antigens: Paraformaldehyde / Saponin Method

Reagents:

• PBS
• PBS-BSA: PBS containing 1% BSA
• 4% (w/v) paraformaldehyde in PBS
• 0.05% (v/v) paraformaldehyde in PBS
• 0.1% (w/v) saponin in PBS

Method

1. Prepare cells appropriately. Refer to Cell Preparation for Flow Cytometry and adjust the cell suspension to a concentration of 1 x 10⁷ cells/ml with cold PBS-BSA.
2. If required, perform staining of cell surface antigens using appropriate directly conjugated mAbs at 4°C. Avoid direct light.
3. Following staining, wash cells once in 3 ml PBS-BSA pellet cells at 400 g and 4°C for 5 minutes. Discard supernatant.
4. Resuspend cells in 100 μl 4% paraformaldehyde/1×10⁶ cells. Incubate for 20 minutes at RT.
5. Wash once in 3 ml PBS-BSA. Pellet cells at 400 g and 4°C for 5 min. Discard supernatant.
6. Resuspend cells in 100 μl 0.1% saponin/1x10⁶ cells. Incubate for 15 minutes at RT.
7. Aliquot 100 μl of cell suspension into the required number of tubes containing directly conjugated antibody at the vendor-recommended dilution. Incubate for at least 30 minutes at 4°C. Avoid direct light.
8. Wash once in 3 ml 0.1% saponin. Pellet cells at 400 g and 4°C for 5 minutes.
9. Resuspend in 200 μl 0.5% paraformaldehyde.
10. Acquire data by flow cytometry.
11. Data analysis.