Antigens from Polyacrylamide Gels:
Antigens may be purified for antibody production using SDS-polyacrylamide gels (SDS-PAGE). Since the antigen is denatured by SDS-PAGE, the resulting antibodies are usually particularly good for techniques that detect antigen in a denatured form (i.e., Western Blotting). However, the resulting antibodies are less likely to bind to native antigen.
Isolating the antigen
The protein sample is run on a preparative gel. Load the gel with enough sample to obtain at least 100-200 μg of antigen per gel slice per injection. The maximum allowable size of the gel slice per injection is 1.5 inches long by 3-4 mm wide by 1.5 mm thick.
Locating the antigen
After electrophoresis, the protein band of interest must be located in the gel, and the gel slice excised for injection. Two methods are suggested. The Side-Strip Method is recommended only for abundant proteins that are well separated. Side-strips are cut from the edge of the gel and stained using typical procedures. The stained side strip is placed back alongside the unstained gel and the protein band excised from the similar location in the unstained gel. A second method, Reversible Staining Method, is generally recommended, particularly when the protein of interest is less abundant and adjacent to other proteins. In this method the entire gel is stained, the relevant band is excised, and then the gel slice is destained (e.g., Pierce, E-zinc reversible stain kit).
Storage and transport of the gel
The excised gel slice should be wrapped in plastic wrap (i.e., saran wrap) along with a few drops of water. Place the gel slice in a secondary container (50 ml tube, plastic bag) along with a moistened paper tissue or towel. The amount of antigen in the gel slice must be given. The maximum allowable size of the gel slice per injection is 1.5 inches long by 3-4 mm wide by 1.5 mm thick. Store the gel at 4°C.
Intracellular Cytokine Staining:
The aim of intracellular cytokine staining is to make use of the rapid flow cytometric analysis of cells to provide both surface phenotype and intracellular cytokine content of single cells within a population of cells responding to antigenic or mitogenic stimulation.
Principle of the test
Stimulated peripheral blood mononuclear cells (PBMCs) are fixed, surface stained and then permeabilized, allowing conjugated antibodies access to proteins within the cell. Prior to fixation, cells are initially subjected to an activation in the presence of Monensin or Brefeldin A (BFA) which inhibit intracellular transport of proteins so cytokines produced during activation will be retained inside the cell. Fixation step minimizes leakage of proteins out of the cell. Permeabilization allows the antibody-fluorochrome conjugate to penetrate and bind to its target cytokine within the cell. Cells are then analyzed on a flow cytometer.
Cell activation and fixation
PBMCs of healthy donors are separated via Ficoll-Paque density-gradient centrifugation. Resuspend PBMCs at 2×106 cells/ml in RPMI-1640 with 10% heat-inactivated fetal calf serum. For stimulation, Phorbol 12-Myristate-13-Acetate (PMA; final concentration 25 ng/ml) with Ionomycin (final concentration 10 μg/ml) is used. Activation is performed in the presence of BFA or Monensin to retain cytokines inside the cells. Then cells are fixed by 1% paraformaldehyde (PFM) in PBS.
Selection of antibodies
To stain for surface proteins of defined cell type, Abs such as CD3, CD4, CD8, etc., are used. The antibody binding activity of these Abs should not be adversely affected by the fixation and permeabilization steps.
Permeabilization: For permeabilization, 0.1% saponin in a balanced salt solution is commonly used. However, It may be advantageous to test other non-ionic detergents (e.g., Triton X100, Nonidet P40 or n-dodecyl-β-glucopyranoside). Pre-made fixation and permeabilization reagents are also commercially available.
Intracellular cytokines staining
Incubation is done in permeabilization medium at 4°C for 30 min with an optimal dose of fluorochrome-conjuguated anti-cytokine antibody. Cell wash is performed in permeabilization buffer. Cells are thoroughly resuspended prior to analysis of the samples by Flow Cytometry. Generally direct staining with antibody-fluorochrome conjugates give the best results, however, indirect staining of unconjugated antibodies with secondary (i.e., anti-Mouse Ig) fluorochrome labelled antibodies has been successful.
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- Assenmacher, M., Schmitz, J. and A. Radbruch. (1994). Flow cytometric determination of cytokines in activated murine Th lymphocytes. Expression of IL-10 in IFNγ and in IL-4 expressing cells. Eur. J. Immunol. 24:1097.
- Prussin, C. & D.D. Metcalfe (1995). Detection of intracytoplasmic cytokines using flow cytometry and directly conjugated anticytokine antibodies. J. Immunol. Methods 188:117.