Detection of Intracellular Antigens by Flow Cytometry

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Introduction

Fix and Perm reagents are designed for use with all commercially available flow cytometers. Alignment and compensation should be performed according to the manufacturer's instructions. Typical staining and scatter patterns are shown below in Figure 1.

Peripheral blood mononuclear cells 
Figure 1. Peripheral blood mononuclear cells stained with FITC-conjugated mouse anti-human myeloperoxidase (MPO). Representative forward (FSC) and side (SSC) scatter patterns and reaction patterns are shown.

Clinical Research Applications: Flow cytometric analysis with monoclonal antibodies has historically been restricted primarily to cell surface molecules. For this reason, intracellular antigens such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulins, etc., were largely excluded from such analysis. Also excluded have been cytometric studies designed to address the cytoplasmic localization of some well established membrane-associated molecules such as CD3 and CD22. Fix and Perm reagents allow intracellular antigen analysis with the equivalent ease as cell surface antigens. The only prerequisite is the availability of suitable antibody conjugates. Most commercially available monoclonal antibody conjugates can be used with the FIX & PERM reagents. However, some determinants are sensitive to the fixation step. This and optimal fixation time may have to be empirically determined for each antibody conjugate.

Materials

Fix & Perm Cell Permeabilization Reagents

Product Code Fixation Medium (A) Permeabilization Medium (B) Tests
GAS-0031 x 5 ml1 x 5 ml50
GAS-0044 x 5 ml4 x 5 ml200
GAS001S-51 x 5 ml 50
GAS001S-1001 x 100 ml 1000
GAS002S-5 1 x 5 ml50
GAS002S-100 1 x 100 ml1000


Materials Required but Not Included:

Phosphate Buffered Saline containing 0.1% NaN 3 and 5% FBS
12 x 75 mm tubes
Centrifuge
Vortex
Sheath Fluid
Pre-cooled Absolute Methanol

Use of  Fix & Perm Reagents:

Fix and Perm reagents are intended for the fixation (Reagent A) and permeabilization (Reagent B) of cells in suspension. This procedure facilitates antibody access to intracellular structures and leaves the morphological scatter characteristics of the cells intact. Specific formulations reduce background staining and allow simultaneous addition of permeabilization medium and fluorochrome-labeled antibodies.

Storage and Stability:

Fix & Perm reagents should be stored and used at room temperature. They are stable for the period shown on the package label when stored as directed. Do not use reagents if a precipitate forms or discoloration occurs. All antibody combinations should be stored at 2-8°C in the dark.

Fix & Perm Kit Components should never be frozen.

Warning:   Reagent A of the FIX & PERM Kit contains formaldehyde which is toxic, allergenic and a suspected carcinogen. Avoid contact with eyes, skin and clothing. All antibodies contain sodium azide as a preservative.

Permeabilization and Staining Procedure


  1. For each sample to be analyzed add appropriate volume of the conjugated antibody directed to the cell surface marker(s) of interest and/or the appropriate isotype control(s) to a 5 ml, 12 x 75 mm tube.

  2. Pipette appropriate volume of adjusted cells (equivalent to 1 x 106 cells) into each tube containing the conjugated antibody or isotype control.

  3. Vortex each tube gently to mix, and incubate for 15 minutes in the dark at room temperature.

  4. Add 100 μl of Reagent A (Fixation Medium) and incubate for 15 minutes at room temperature.

  5. Wash once in 3 ml PBS + 0.1% NaN3 + 5% FBS.

  6. Centrifuge for 5 minutes at 300-350 x g, aspirate the supernatant, and vortex to fully resuspend the cell pellet.

  7. Add 100 μl of Reagent B (Permeabilization Medium) and the recommended volume of the FITC- and/or PE- conjugated intracellular antibody or the corresponding isotype control.

  8. Vortex 1-2 seconds and incubate for 20 minutes.

  9. Wash once in 3 ml PBS + 0.1% NaN3 + 5% FBS.

  10. Centrifuge for 5 minutes at 300-350 x g and aspirate the supernatant.

  11. Resuspend cells in sheath fluid for immediate analysis or in 0.5 ml of 0.1% paraformaldehyde fixative solution for storage at 2-8ºC in the dark. Fixed cells should be analyzed within 24 hours.

A modification of this protocol using precooled absolute methanol has been shown to give better results for certain cell cycle antigens such as BrdU, Ki-67, and PCNA when using FITC-conjugated antibodies. This modification is not recommended when using PE-conjugated antibodies.

Methanol Modification

  1. For each sample to be analyzed, add 100 μl of adjusted cell volume (equivalent to 1 x 106 cells) to an appropriate 5 ml, 12  x 75 mm tube.

  2. Add 100 μl of Reagent A (Fixation Medium) and incubate for 2–3 minutes at room temperature.

  3. Add 4 ml of pre-cooled absolute methanol (0–4°C) and vortex.

  4. Incubate for an additional 10 minutes at 0–4°C.

  5. Centrifuge for 5 minutes at 300–350 x g and wash with wash medium PBS + 0.1% NaN3 + 5% FBS.

  6. Add 100 μl of Reagent B (Permeabilization Medium) and appropriate volume of intracellular antibody(ies) or corresponding isotype control(s).

  7. Vortex at low speed for 1–2 seconds and incubate for 30 minutes at room temperature.

  8. Wash once in 3 ml wash medium (PBS + 0.1% NaN3 + 5% FBS).

  9. Centrifuge for 5 minutes at 300–350 x g and aspirate the supernatant.

  10. Resuspend cells in sheath fluid for immediate analysis, or fix in 0.5 ml of 0.1% paraformaldehyde and store at 2–8°C in the dark. Fixed cells should be analyzed within 18 hours.

References

  1. Knapp, W., O. Majdic, and H. Strobl. 1993. Flow cytometric analysis of intracellular myeloperoxidase and lactoferrin in leukemia diagnosis. Recent Results Cancer Res. 131: 31-40.

  2. Strobl, H., M. Takimoto, O. Majdic, G. Fritsch, C. Scheinecker, Höcker and W. Knapp. 1993. Myeloperoxidase expression in CD34+ normal human hemopoietic cells. Blood 82: 2069-2078.

  3. Murao, S. I., F. J. Stevens, A. Ito, and E. Huberman. 1988. Myeloperoxidase: a myeloid cell nuclear antigen with DNA-binding properties. Proc. Nat. Acad. Sci. USA 85: 1232-1236.

  4. Koeffler, H. P., J. Ranyard, and M. Pertcheck. 1985. Myeloperoxidase: its structure and expression during myeloid differentiation. Blood 65: 484-491.

  5. Campana, D., J. S. Thompson, P. Amiot, S. Brown, and G. Janossy. 1987. The cytoplasmic expression of CD3 antigens in normal and malignant cells of the T lymphoid lineage. J. Immunol. 138: 648-655.

  6. Janossy, G, E. Coustan-Smith, and D. Campana. 1989. The reliability of cytoplasmic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia: A study of 500 Cases. Leukemia 3: 170-181.
L12001      3-Dec-2003