FlowCytometryAnalysis

Purpose

Flow cytometry employs instrumentation that scans single cells flowing past excitation sources in a liquid medium. The technology can provide rapid, quantitative, multiparameter analyses on single living (or dead) cells based on the measurement of visible and fluorescent light emission. Flow cytometry is a widely used method for characterizing and separating individual cells. This basic protocol focuses on: measure fluorescence intensity produced by fluorescent-labled antibodies and ligands that bind specific cell-associated molecules.

Material

• Staining solution: L-15 medium (Sigma catalog L-99002) plus 2.5% FCS (4^oC)

• Fluorescent-labeled antibodies

• 5 ml polystyrene tube

• 96 well V-bottom plate

• Propidium iodide solution

• Fixation solution: 2% paraformaldehyde in PBS (4^oC)

• Standard fluorescent beads

• Becton Dickinson FACScan

• CELLQuest Software

Procedure

Immunofluorescence Staining 

The Staining procedure is carried out at 4^oC and dark. Appropriate controls are needed to get correct results, such as unstained control, isotype antibody stained control and positive control etc.

1. Make single cell suspension in L-15 at a concentration of 1-2x10⁷ cells/ml

2. Add 50ul cell suspension to 5ml polystyrene tube (or 96 well V-bottom palte)

3. Add 50ul of appropriately diluted labeled antibody to the cells and mix gentlely

4. Incubate at 4^oC in dark for 30 minutes

5. Wash cells by adding 2ml L-15 and centrifuge 5 minutes at 1000rpm (300xg)
   For 96 well plate, wash cells three times with 150ul L-15

6. Resuspend stained cell pellets in 400ul L-15 at 4^oC for flow cytomery

7. Cells can be resuspended in fixation solution and stored for 1 week (optional)

8. Add 10ul propidium iodide prior to analysis to detect dead cells (optional)

9. Calibrate the FACScan by standard fluorescent beads (optional)

Flow Cytometry Analysis

Flow cytometers are complex instruments that require a well-trained operator. Prior to use the Becton Dickinson FACScan, take a training course with an experienced user. Follow the rules posted in the working area for appropriate maintenance of the instrument.

1. Turn on the FACScan power and wait until the indicator light changes from NOT READY to STANDBY. Restart the computer to connect with theFACScan.

2. Open your account on the desktop and use the appropriate template for aquiring data (see reference for making correct setting).

3. Turn fluid control valve to RUN and place sample tube on FACScan

4. Analysis data with CELLQuest Software

References

1. Becton Dickinson Immunocytometry Systems

2. Current Protocols in Immunology