Technical Information

Technical Information

Fluorescence Protocol 2.0

Materials


Cytokine-specific Primary Antibodies
  • unlabeled or biotinylated antigen-affinity purified polyclonal antibodies (R&D Systems 'AF' or 'BAF' series) or selected monoclonal antibodies.
Secondary Antibodies and Secondary Reagents
  • Biotinylated donkey anti-goat IgG (Jackson Immuno Research Lab Catalog # 705-066-147)
  • Biotinylated goat anti-mouse IgG1 (Caltag Lab Catalog # M32115)
  • FITC-labeled goat anti-mouse IgG1 (Caltag Lab Catalog # M32001)
  • FITC-labeled anti-mouse IgG2A (Caltag Lab Catalog # M32201)
  • FITC-labeled anti-mouse IgG2B (Caltag Lab Catalog # M32401)
  • Oregon Green coupled anti-mouse IgG (Molecular Probes Catalog # 06380)
  • Oregon Green-avidin D(Molecular Probes Catalog # A6374)
  • FITC-Avidin D (Sigma Catalog # A-2001)
Buffers and Additional Supplies
  • Fixation Buffer Formaldehyde (37% v/v Sigma) is diluted in PBS to a final formaldehyde concentration of 2% (v/v) and adjust pH to 7.4. Light sensitive, store at 4°C in the dark; prepare working dilution just prior to fixation
  • Wash Buffer Earls Buffered Salt Solution (EBSS) (Gibco BRL)
  • Wash Buffer-Saponin EBSS with 0.1% (w/v) Saponin (Sigma Catalog # S4521) and adjust pH to 7.2-7.4 with NaOH.
  • Endogenous Peroxidase Blocking Buffer 3mM NaN3 in EBSS with 1% H2O2 and 0.1% (w/v) Saponin.
  • Endogenous Biotin Blocking Buffer Avidin/Biotin Blocking Kit (Vector Lab Catalog # SP2001). Both Avidin D and Biotin should be supplemented with 0.1% (w/v) Saponin.
  • Fluorescence Anti Fading Mounting Medium anti-fade mounting medium i-BRITE Plus (SF40000, Neuromics, Inc.)
  • Saponin stock Prepare a stock of 10 % (w/v) Saponin in EBSS. Prepare fresh, since crude saponin powder batches are generally fungi infected.
  • Intracellular transport inhibitor Brefeldin A (Sigma Catalog # B7651)
  • Microscope slides Adhesion slides (smeared cells); TC microscope glass slides (tissue sections); HTC slides (Cel-line Assoc. Catalog # 10-618)
  • Frozen tissue embedding OCT-compound (VWR Catalog # 25608-930)
  • Humidified Chamber A plastic light protected box lined with wet paper towels.

Controls


Positive staining controls
  • Stain fixed, permeabilized cytokine-cDNA transfected eukaryotic cells expressing the intra-cellular target cytokine protein.
  • Stain cultured blood mononuclear cells or spleen cells that have been harvested, fixed and permeabilized at the peak of cytokine production after in vitro stimulation with strong polyclonal activators such as phorbol 12-myristate 13 acetate/ionomycin, anti-CD3+CD28 monoclonal antibody, LPS or bacterial superantigens such as staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB) or streptococcal pyrogenic exotoxin A (SPE-A).
Negative staining controls
  • Ligand blocking control: to study the specificity of the cytokine staining by a preincubation of the cytokine-specific antibody with its target cytokine at a molar ratio of 1:10 prior to addition to the cells.
  • Isotype matched control immunoglobulin: Stain the cells by a primary isotype pre-matched immunoglobulin of irrelevant antigen-specificity at the same concentration as the cytokine-specific antibodies.
  • Unlabeled antibody control: Cells that will be stained by a fluorochrome or a biotinylated primary cytokine-specific antibody will be incubated initially with the unconjugated version of the same antibody.
  • Non transfected eukaryotic cells and unstimulated PBMNC are good negative controls.

Immunofluorescent staining of cytokine-producing cells in suspension

Cells intended for analysis by flow cytometry should be cultured in vitro prior to harvest in the presence of an intracellular transport inhibitor such as Brefeldin A (1-5 µg/mL). This step will increase the concentration of intracellular cytokines, which will enhance the discrimination of cytokine-positive cells from background signals caused by autofluorescence. The treatment will, however, ruin the characteristic morphology, seen in the UV-microscope, of a rounded perinuclear dot generated by the the accumulation of the cytokines in the Golgi organelle.

Computerized image analysis offers an alternative to score these cells by automated technique without any need for cell cultures with Brefeldin A. The computer program is designed in such a way that the system will record single cells as positive or negative cytokine producers based on the intensity and the color of the cytokine staining combined with an appreciation of the characteristic morphology of the juxtanuclear staining pattern generated by the accumulation of the cytokines in the Golgi organelle.

Sample Preparation and Fixation

  1. Harvest cells and wash twice in V-bottomed tubes with cold Wash Buffer by centrifugation (400 x g for 5 minutes) at 4°C to remove extracellular cytokines.
  2. Add 0.5 mL of the Fixation Buffer to the cell pellet and incubate for 20 minutes.
  3. Note: During the fixation time, the cells are vortexed to avoid cell aggregation.
  4. Wash the cells twice in Wash Buffer Saponin by centrifugation (400 x g for 5 minutes).
  5. For cytokine staining aliquot cells at a concentration of 1-5 x 106/mL in tubes or microplates. Fixed cells can also be frozen in culture medium supplemented with 15% fetal bovine serum and 10% dimethylsulfoxide (DMSO) for future cytokine staining.

Antibody Incubation

All antibody incubations and washes should be performed in Wash Buffer-Saponin to keep the cells permeable for antibodies to penetrate the cell membranes.

  • Detection using Biotin-labeled antibodies
    1. Block endogenous biotin activity with the Avidin/Biotin blocking kit in a two step procedure for 30 minutes in the presence of saponin, described in steps 2-4 below.
    2. Incubate cells with Avidin for 15 minutes supplemented with saponin (0.1% w/v).
    3. Wash the cells twice in Wash Buffer-Saponin by centrifugation (400 x g for 5 minutes).
    4. Incubate the cells in Biotin for 15 minutes supplemented with saponin (0.1% w/v).
    5. Wash the cells twice in Wash Buffer-Saponin by centrifugation (400 x g for 5 minutes).
    6. Add 50 µL of unlabeled or biotinylated cytokine-specific antibodies (0.5-5 µg/mL) diluted in Wash Buffer-Saponin to the vortexed cell pellet.
    7. Incubate for 30 minutes at room temperature.
    8. Wash twice in Wash Buffer-Saponin by centrifugation (400 x g for 5 minutes).
    9. Incubate the cells for 30 minutes at room temperature with 50 µL of a biotinylated secondary antibody (either biotin-donkey anti-goat IgG Fab2 diluted 1:700 or biotin-goat anti-mouse IgG1 or IgG2A or IgG2B diluted 1:500) in Wash Buffer-Saponin.
    10. Wash the cells twice in Wash Buffer-Saponin.
    11. Incubate the centrifuged cell pellet for 30 minutes at room temperature with 50 µL of Oregon-Green-Avidin D or FITC-Avidin D (2-5 µg/mL Wash Buffer-Saponin).
    12. Wash twice in Wash Buffer-Saponin by centrifugation (400 x g for 5 minutes) followed by a final wash in Wash Buffer by centrifugation without any saponin (400 x g for 5 minutes). Cells are then ready for analysis by flow cytometry or alternatively by UV-microscopy.
    13. For UV microscopy analysis the cells can be resuspended in 0.1 M sodium citrate and drops of the suspension can be put on glass slides and be left to air-dry. The dried cell preparations can be mounted in Anti-Fading Buffer to reduce UV quenching of FITC. Oregon-Green does not fade and can stand daylight without problem. In addition it gives a very crisp and intensive signal. Stained cells can be stored at 4°C for several weeks without loss of staining quality. It is possible to perform multiple color-staining in the same cells using different fluorochromes by using this staining protocol sequentially. Between staining for different antigens, block with the Biotin/Avidin kit to prevent binding to biotinylated antibodies from the previous staining.
  • Detection using fluorochrome-labeled antibodies
    If cytokine producing cells are to be detected by fluorochrome-labeled primary or secondary antibodies there is no need to block endogenous biotin activity. Background signals are often reduced when fluorochrome-labeled antibodies are used in Wash Buffer-Saponin supplemented with 5% human AB serum.
    1. Incubate the suspended cells for 30 minutes at room temperature with 50 µL of unlabeled or fluorochome-labeled primary cytokine-specific antibodies (0.5-5 µg/mL) diluted in Wash Buffer-Saponin supplemented with 5% human AB serum.
    2. Wash the cells twice by centrifugation in Wash Buffer-Saponin (400 x g for 5 minutes).
    3. Incubate cells for 30 minutes at room temperature with 15 µL of a fluorochrome-labeled secondary antibody (either FITC-labeled anti-mouse IgG1, IgG2A or IgG2B diluted 1:300) in Wash Buffer-Saponin supplemented with 5% human AB serum.
    4. Cells are ready for analysis by flow cytometry or alternatively by UV-microscopy.

Immunostaining of individual cytokine-producing cells smeared on slides


Suitable for use on single-cell suspensions from peripheral blood, lymphoid tissue or cultured cell-lines.

Sample Preparation and Fixation

  1. Harvest cells and wash twice in V-bottomed tubes with cold Wash Buffer by centrifugation (400 x g for 5 minutes) to remove extracellular proteins, including cytokines.
  2. Resuspend to 1-5 x 106 cells/mL in Wash Buffer.
  3. Transfer 10-15 µL of the cell suspension to each reaction field on the adhesion slide.
  4. Allow the cells to adhere electrostatically in a monolayer for 10 minutes at room temperature in the humidified chamber to prevent the cells from drying out.
  5. Add 50 µL of ice-cold Fixation Buffer to each field to fix the cells.
  6. Incubate for 20 minutes at 4°C.
  7. Wash three times with Wash Buffer to remove free formaldehyde.
  8. Add 25 µL of 2% fetal bovine serum in Wash Buffer to block unbound surface area on the slide.
  9. Incubate for 10 minutes at 37°C.
    1. Wash slides three times in Wash Buffer-Saponin. The slides are now ready for staining.
    2. Alternatively, wash the slides in Wash Buffer and allow the slides to dry. Dried slides can be stored at -20°C several months before being stained. Prior to staining the slides should be washed in Wash Buffer-Saponin.

Antibody Incubation

All antibody incubations and washes are performed in Wash Buffer-Saponin to keep the cells permeable for antibodies to penetrate the cell membranes.

  1. Detection using Biotin-labeled antibodies
    1. Incubate in Endogenous Peroxidase Blocking Buffer for 30 minutes at room temperature in the dark to block endogenous peroxidase activity in the cells (this step can be omitted if cells are to be stained by fluorochromes or non-peroxidase based enzymatic methods).
    2. Block endogenous biotin activity with the Avidin/Biotin blocking kit in a two step procedure for 30 minutes in the presence of saponin, described in steps 3-5.
    3. Incubate each cell spot on slides with Avidin for 15 minutes supplemented with saponin (0.1% w/v).
    4. Wash each cell spot on slides twice in Wash Buffer-Saponin.
    5. Incubate in Biotin for 15 minutes supplemented with saponin (0.1% w/v).
    6. Wash each cell spot on slides twice in Wash Buffer-Saponin.
    7. Incubate each cell spot on slides for 30 minutes at room temperature with 15 µL unlabeled or biotinylated cytokine-specific antibodies (0.5-5 µg/mL) diluted in Wash Buffer-Saponin.
    8. Wash slides three times in Wash Buffer-Saponin.
      Note: If using R&D Systems biotinylated antibody skip steps 9 and 10 and continue.
    9. Incubate each cell spot on slides for 30 minutes at room temperature with 15 µL of a biotinylated secondary antibody (either biotin-donkey anti-goat IgG Fab2 diluted 1:700; or biotin-goat anti mouse IgG1 or IgG2A or IgG2B diluted 1:500) in Wash Buffer-Saponin.
    10. Wash slides three times in Wash Buffer-Saponin.

Cytokine-specific staining based on either biotinylated primary antibodies or unlabeled primary antibodies along with biotinylated secondary antibodies can then be developed by techniques based on immunoflourescence or immunoenzymatic methods.

Immunofluorescent Technique

  1. Incubate slides for 30 minutes at room temperature with 15 µL of Oregon-Green Avidin or FITC-Avidin at a concentration of 2-5 µg/mL in Wash Buffer-Saponin.
  2. Wash slides twice in Wash Buffer-Saponin.
  3. Wash slides once in Wash Buffer only. Allow slides to air dry before mounting in Mounting Buffer. FITC staining, but not Oregon-Green staining, will require a mounting medium including some anti-fading substance to reduce UV quenching. It is possible to store fluorochrome-stained slides for long periods in the freezer, provided that they have not been mounted in Mounting medium.
  1. Detection using fluorochrome-labeled antibodies
    If cytokine producing cells are to be detected by fluorochrome-labeled primary or secondary antibodies there is no need to block endogenous peroxidase or biotin activity. Background signals are often reduced when fluorochrome-labeled antibodies are used in Wash Buffer-Saponin supplemented with 5% human AB serum.
    1. Incubate each cell spot on slides for 30 minutes at room temperature with 15 µL of either unlabeled or fluorochome-labeled primary cytokine-specific antibodies (0.5-5 µg/mL) diluted in Wash Buffer-Saponin supplemented with 5% human AB serum.
    2. Wash slides three times in Wash Buffer-Saponin.
    3. Incubate cells for 30 minutes at room temperature with 15 µL of flurochrome-labeled secondary antibody (either FITC-labeled anti-mouse IgG1 or IgG2A or IgG2B diluted 1:300) in Wash Buffer-Saponin supplemented with 5% human AB serum.
    4. Wash slides three times in Wash Buffer-Saponin and air dry the slides.
    5. Mount and coverslip with Fluorescence Anti Fading Mounting Medium.

Frequently Asked Questions


Question: Is the cytokine staining specific?
Test: Incubate cytokine-detecting antibody with target cytokine overnight and finally add 0.1 (saponin prior to staining as in Question 4.
Cause of Background: Cytokine-detecting antibody used at too high concentration or unsuitable for immunostaining.
Remedy: Titrate antibody or try another cytokine detecting antibody.

Technical Hints


Permeabilization:

It is crucial that saponin is present during all antibody incubations and washes to make the staining procedure successful. For detection of intracellular cytokines, the cytokine-specific antibodies must penetrate through the cell surface membrane, the cytosol, the membranes of the endoplasmic reticulum and the Golgi organelle. The detergent, saponin has been shown to intercalate in the membranes to replace cholesterol and to permeabilize cells in a reversible way, maintaining much of the morphology of the membrane structure of the cell.

Fixation:

A solution of phosphate-buffered formaldehyde has been found to preserve cell morphology as well as surface and intracellular antigenicity with minuscule cell aggregation and cell loss. Only fixed cells will stand the effects of detergent treatment.

Controls:

Evidence for specificity of the cytokine staining should be based on parallel studies of isotype controls, staining with the secondary antibodies alone and an abolishment of immunoreactivity by preabsorption of the cytokine-specific antibody with the corresponding cytokine protein.

Stimulation of PBMNC for cytokine production:

The strongest and most diversified cytokine production is seen after PMA-ionomycin activation of Ficoll separated PBMNC. Both monocytes and lymphocytes will be activated. The cells are co-cultivated with PMA (1 ng/mL), ionomycin (500 nM) and harvested after 4 hours. After 4 hours, one can expect to see IL-1a, IL-1ß, IL-1ra, IL-2, IL-3, for various periods. If cells are harvested after 3 hours, one can find monokines such as IL-1a. However, cytokines produced by lymphocytes, IL-12 or substantial IL-10 production is not found in the cultures.

References

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  2. Litton, M. et al. (1997) Human Cytokine Protocols, Debets/Savelkoul eds., Humana Press, USA, In press.
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Our thanks to Drs. Ulf Andersson, Ann-Charlotte Aveberger, Lars Björk, Caroline Ekberg, Thomas Fehniger, Mark Litton, Lena Radler, Birgitta Sander, Ulrika Skansén-Saphir, Ann-Kristin Ulfgren, Karin Ågren, Karin-Åkerlund (Department of Immunology, Stockholm University) for the submission of the protocol and revision of the manuscript.