Flow Cytometry Analysis
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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 (4o C)
- Fluorescent-labeled antibodies
- 5 ml polystyrene tube
- 96 well V-bottom plate
- Propidium iodide solution
- Fixation solution: 2% paraformaldehyde in PBS (4o C)
- Standard fluorescent beads
- Becton Dickinson FACScan
- CELLQuest Software
Procedure
Immunofluorescence Staining
The Staining procedure is carried out at 4o C and dark. Appropriate controls are needed to get correct results, such as unstained control, isotype antibody stained control and positive control etc.
- Make single cell suspension in L-15 at a concentration of 1-2x107 cells/ml
- Add 50ul cell suspension to 5ml polystyrene tube (or 96 well V-bottom palte)
- Add 50ul of appropriately diluted labeled antibody to the cells and mix gentlely
- Incubate at 4o C in dark for 30 minutes
-
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 - Resuspend stained cell pellets in 400ul L-15 at 4o C for flow cytomery
- Cells can be resuspended in fixation solution and stored for 1 week (optional)
- Add 10ul propidium iodide prior to analysis to detect dead cells (optional)
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.
- Turn on the FACScan power and wait until the indicator light changes from NOT READY to STANDBY. Restart the computer to connect with theFACScan.
- Open your account on the desktop and use the appropriate template for aquiring data (see reference for making correct setting).
- Turn fluid control valve to RUN and place sample tube on FACScan
- Analysis data with CELLQuest Software
References
- Becton Dickinson Immunocytometry Systems
- Current Protocols in Immunology