Indirect Staining Mononuclear Cells

Indirect Immunofluorescence Staining of Mononuclear Cells

Scope

The indirect method is used to enhance the fluorescence signal and also to facilitate multicolor staining of human cells when direct conjugated reagents are not available. 1,2 BD offers two methods for indirect staining:

• biotin-avidin (or streptavidin)
• conventional second antibody

In the biotin-avidin method, cells are incubated first with biotin-conjugated monoclonal antibody and then subsequently incubated with fluorochrome-conjugated avidin. In the second antibody method, cells are incubated with an unconjugated monoclonal antibody followed by fluorochrome-conjugated goat anti-mouse Ig.

These sandwich techniques provide increased fluorescence intensity, which is valuable for microscopy. However, indirect methods can result in the formation of complexes that will artifactually stain Fc receptor-binding cells. The biotin-avidin method gives the least amount of artifactual staining particularly when streptavidin conjugates are used. Indirect methods will alter the proportionality between the amount of antigen and the fluorescence intensity per cell, therefore, these methods are not recommended for assessing the absolute number of antigenic determinants per cell. However, indirect methods can be used to determine the relative brightness differences between cell populations.

Reagents and Equipment

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 disposable 12 x 75-mm capped polystyrene test tubes (BD Cat. No. 2058) or equivalent.
3. Optional: Falcon 96-well U-bottom microtiter plates (BD Cat. No. 353918) or equivalent.
4. Micropipettor with tips (BD Electronic Pipette, BD Cat. No. 343246 or equivalent).
5. BD biotin-conjugated monoclonal antibody to human surface antigen; unconjugated monoclonal antibody.
6. Wash buffer: phosphate-buffered saline (PBS) containing 0.1% sodium azide and 2% fetal bovine serum (FBS). Store at 2° to 8°C. Do not use PBS containing biotin with the biotin-avidin indirect method.
   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 to avoid deposits in lead or copper plumbing where explosive conditions can develop.
7. BD fluorochrome-conjugated avidin or fluorochrome- conjugated streptavidin monoclonal antibody; fluorochrome-conjugated goat anti-mouse Ig monoclonal antibody
8. Centrifuge
9. 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.
10. FACS brand flow cytometer. Refer to the appropriate instrument user’s guide for information.

Procedure

Specimen Collection and Preparation

Collect blood aseptically by venipuncture1,2 into VACUTAINER CPTs containing sodium heparin. Follow the manufacturer’s collection guidelines for the minimum volume of blood to be collected. Before storage, centrifuge CPTs and resuspend peripheral blood mononuclear cells (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 densitygradient 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. Dilute unconjugated or biotin-conjugated monoclonal antibody by adding 20 of μL antibody to 30 μL of wash buffer for each test. Add the 50 μL of diluted antibody to microtiter wells or 12 x 75-mm tubes.
2. Adjust concentration of the cell suspension to 2 x 10 7 cells/mL of wash buffer. 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. 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.
   For staining in tubes: Incubate the mixture for 30 to 45 minutes on ice. Add 2 mL of cold wash buffer, and centrifuge at 300 x g for 5 minutes at 2° to 8°C. Remove the supernatant.
5. Dilute the appropriate second-step reagent (fluorochrome-conjugated avidin or goat anti-mouse Ig) in wash buffer according to the instructions provided on the appropriate second-step reagent data sheet.
6. For staining in microtiter plates: Add the second-step mixture to the wells and incubate 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: Add the second-step mixture to the tubes and incubate 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.
7. Store at 2° to 8°C until analyzed.
8. Analyze on a FACS brand flow cytometer. Mix samples thoroughly before acquisition.