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Blizard Institute - Faculty of Medicine and Dentistry
  1. Blizard Institute
  2. Research
  3. Core Facilities
  4. Flow Cytometry
  5. Uses of Flow Cytometry
  6. Multicolour Analysis
  7. Cytoplasmic antigen analysis

Cytoplasmic antigen analysis

To detect intracellular antigens it is necessary to permeabilise the cell. Many reagents are available commercially to facilitate this.

 

  • Commercial permeabilisation reagents [PDF - 52KB]

There is a wide variety of different types of intracellular antigen that can be detected by flow cytometry, including:-

Cytokines -Th1Th2 responses

Lymphocytes and splenocytes involved in inflammatory or allergic responses produce a preponderance of IFNgamma or IL-4 respectively. KO murine splenocytes Th1Th2 responses were compared to wild type splenocytes. The KO showed an increased capacity to produce IFNgamma compared to the wild type response whilst IL-2 and IL-4 levels remained the same as the wild type.

Detection of intracellular IL-17

Interleukins (e.g. IL1-17) can be measured intracellularly in lymphocytes after stimulation typically with PMA/ionomycin and blocking the Golgi apparatus with the antibiotic Brefeldin A or Monensin, see protocol. 

Activation of whole blood

  1. Dilute whole blood 1:1 volume to volume (e.g. 100 μl:100 μl) with RPMI1640 medium and mix well.
  2. Add cell activator or mitogen to the diluted blood e.g. 50 ng/ml PMA + 1μg/ml calcium ionophore or PMA + 1 μM ionomycin (final concentration) in the presence of a protein calcium inhibitor such as Golgi plug containing brefeldin A ( ng/ml).
  3. Vortex briefly to mix. Aliquot 200μl into 12 x 75 plastic tubes. Incubate for 4-6 h in 5% CO2 at 37˚ C.
  4. Add 2ml of Pharmlyse (eBioScience), vortex and incubate for 10 min at RT in the dark.
  5. Spin 5 min, 500g.
  6. Aspirate the supernatant, Wash 1X in staining buffer. Spin 5 min at 500g. Aspirate the supernatant.
  7. Continue with staining after adding 50μl PBS to each tube.

Transcription factors

Foxp3 the self-reactive transcription factor, the master regulator involved in lymphocyte development and function of regulatory T cells can be measured intracellularly by flow cytometry, see protocol. 

Foxp3 Intracellular staining in whole blood

  1. To 100 μl whole blood add anti mouse CD3FITC, CD4APC, CD25PEor mouse isotypePE (1 mg/ml). Mix on Vortex  and incubate for 20 min at RT.
  2. Add 2ml of Pharmlyse (Becton Dickinson), vortex and incubate for 10 min at RT in the dark.
  3. Spin 5 min, 500g.
  4. Aspirate the supernatant.
  5. Add 100 ml of Fix solution A (Caltag Fix and Perm Kit), mix on vortex and incubate for 15 min at RT.
  6. Add PBS and spin 5 min, 500g.
  7. Aspirate the supernatant.
  8. Add 100 ml of Perm solution B  (Caltag Fix and Perm Kit). Mix on Vortex and incubate for 15 min at RT.
  9. Add PBS and spin 5 min, 500g.
  10. Aspirate the supernatant.
  11. Add anti mouse Foxp3PB(0.5 mg) or mouse isotypePB (0.5 mg) to cell pellets. Mix on Vortex and incubate for 30 min on ice.
  12. Add PBS and spin 5 min, 500g.
  13. Aspirate the supernatant.
  14. Add PBS <0.5ml.
  15. Collect >25,000 events.

Cyclins

The intracellular detection of cyclins can be combined with cell cycle analysis, see cell cycle analysis section.

Apoptotic factors

The pro-apoptotic factor, bak undergoes a conformational change when activated during apoptosis involving the mitochondria. The degree of bak activation can be determined intra- cellularly by use of mcab that detects the activated form of bak, see protocol. 

Pro-Apoptotic Factor Intracellular Labelling Protocol

  1. Pelleted cells fixed with 0.25% paraformaldehdye for 1 hour on ice.
  2. Cells were washed in PBS.
  3. Primary mcab (1 mg) was added to cell pellets permeabilised in 0.01% saponin (Sigma) and incubated on ice for 30 mins.
  4. Cells were washed in PBS/0.01% Saponin.
  5. Secondary antibody (0.5 mg) were added to cell pellets and incubated for 30 mins on ice.
  6. Cells were washed in PBS/0.01% Saponin and resuspended in 0.5 ml PBS.
  7. At least 10,000 events were analysed by flow cytometry.
  • cytokines e.g. IL-17
  • apoptotic factors e.g. bak activation
  • cyclins
  • Transcription Factors e.g. Foxp3
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