Protocol Direct Immunofluorscence Staining

Direct Immunofluorescence Staining of Mononuclear Cells

Scope

Use this method to detect cells bearing specific membrane antigens. Begin by adding peripheral blood mononuclear cells (PBMCs) to fluorochrome-conjugated monoclonal antibodies that bind specifically to cell surface antigens. Next, wash the stained sample to remove excess antibody and debris. Analyze the cells by flow cytometry.

Reagents and Equipment Required

  1. VACUTAINER Cell Preparation Tubes (CPTs) (BD Cat. No. 362753) or Ficoll-Paque separation medium. Refer to the Ficoll-Paque package insert for materials and reagents required.
  2. Falcon 15-mL conical tubes (BD Cat. No. 2099) or equivalent.
  3. Falcon disposable 12 x 75-mm capped polystyrene test tubes (BD Cat. No. 2058) or equivalent.
  4. Optional: Falcon 96-well U-bottom microtiter plates (BD Cat. No. 353918) or equivalent.
  5. Micropipettor with tips (BD Electronic Pipette, BD Cat. No. 343246) or equivalent.
  6. BD fluorochrome-conjugated monoclonal antibodies to human cell surface antigens. Refer to the appropriate reagent package insert for more information.
  7. Centrifuge
  8. Wash buffer: phosphate-buffered saline (PBS) containing 0.1% sodium azide (Dulbecco’s PBS without calcium, magnesium, or phenol red,pH 7.2 ± 0.2). Filter the PBS through a 0.2-μm filter prior to use; store at 2° to 8°C.
    WARNING: Sodium azide is harmful if swallowed. Keep out of reach of children. Keep away from food, drink, and animal feedingstuff. Wear suitable protective clothing. If swallowed, seek medical advice immediately and show the container or label. Contact with acids liberates very toxic gas. Azide compounds should be flushed with large volumes of water during disposal toavoid deposits in lead or copper plumbing where explosive conditions can develop.
  9. Staining buffer: PBS containing 0.1% sodium azide and 2% fetal bovine serum (FBS). Store at 2° to 8°C
  10. 1% paraformaldehyde solution prepared in PBS containing 0.1% sodium azide. Store at 2° to 8°C in amber glass for up to 1 week.

    WARNING:Formaldehyde is harmful by inhalation, in contact with skin, and if swallowed. It is irritating to eyes and skin. Exposure can cause cancer. Possible risks of irreversible effects. Can cause sensitization by skin contact. Keep locked up and out of the reach of children. Keep away from food, drink, and animal feedingstuff. Wear suitable protective clothing and gloves. If swallowed, seek medical advice immediately and show the container or label. Dispose of according to federal, state, and local regulations.
  11. FACS brand flow cytometer. Refer to the appropriate instrument user’s guide for information.

Procedure

Specimen Collection and Preparation

Collect blood aseptically by venipuncture 1,2 into a VACUTAINER CPT containing sodium heparin. Follow the manufacturer’s collection guidelines for the minimum volume of blood to be collected. Before storage, centrifuge CPTs and resuspend PBMCs in the autologous plasma by gently inverting each tube several times. Store each CPT at room temperature (20° to 25°C) on its side. Use within 24 hours of collection. PBMCs can also be separated from whole blood by Ficoll-Paque density-gradient centrifugation.

WARNING: All biological specimens and materials with which they come into contact should be handled as if capable of transmitting infection and disposed of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Avoid specimen contact with skin and mucous membranes.

  1. Transfer cell suspension to 15-mL conical tubes. Wash with wash buffer.
  2. Resuspend cells in staining buffer and adjust concentration of cell suspension to 2 x 10 7 cells/mL. Cells should be >90% viable.
  3. Add 50 μL of the cell suspension (1 x 10 6 cells) to each microtiter plate well or to each 12 x 75-mm tube.
  4. Add appropriate volume of fluorochrome-conjugated monoclonal antibody.
  5. For staining in microtiter plates: Incubate the mixture for 30 to 45 minutes on ice. Centrifuge at 200 x g for 3 minutes at 2° to 8°C. Remove the supernatant. Wash two times with 100 μL of cold wash buffer. Centrifuge after each washing at 200 x g for 3 minutes. Remove the supernatant. Resuspend cells in 200 μL of 1% paraformaldehyde solution and transfer samples to 12 x 75-mm tubes containing 300 μL of 1% paraformaldehyde solution.
    For staining in tubes: Incubate the mixture for 30 to 45 minutes on ice. Add 2 mL of cold wash buffer. Centrifuge at 300 x g for 5 minutes at 2° to 8°C. Remove the supernatant. Resuspend cells in 0.5 mL of 1% paraformaldehyde solution to approximately 1 x 10 6 cells/mL.
  6. Store at 2° to 8°C until analyzed.
  7. Analyze on a FACS brand flow cytometer. Mix samples thoroughly before acquisition.

References

  1. Clinical Applications of Flow Cytometry: Quality Assurance and Immunophenotyping of Peripheral Blood Lymphocytes; Tentative Guideline. Villanova, PA: National Committee for Clinical Laboratory Standards; 1992. NCCLS document H42-T.
  2. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture: Approved Standard. Villanova, PA: National Committee for Clinical Laboratory Standards; 1991. NCCLS document h2-A3.

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