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Flowcytometry Tips

Sample preparation:

1. Be sure that all the samples used for flow cytometry are single cell suspension in case of block of apparatus by cell aggregates and fragments.

2. The concentration of single cell suspension should reach 1X106/ml, or too low concentration may have bad effect on the results.

3. Suitable methods should be made for different samples to be disposed in order to obtain single cell suspension, such as mechanically disjointed, enzymatic discomposition, and so on.

4. Avoid death cells producing by personal equation. If the content of death cells > 20%, then the results would be unreliable.

Fluorescence staining:

1. The antibody coupled with fluorescence dyes should be preserved at 4℃ in dark.

2. Blocking reagents such as 0.5% BSA and 1% fetal bovine serum should be used to block non-specific binding sites.

3. The sample stained by fluorescence dyes should be kept away from lights to make sure its stability.

4. The buffer used for washing cells after staining should also be added with 0.5% BSA and 1% fetal bovine serum, not only for blocking non-specific binding sites, but also for maintain the activity of cells.

5. Adequate washing should be done to avoid false-positive and false-negative.

6. Intese mixing should be avoided in case of producing too many broken cells which may result of non-specific flurescence.

7. Suitable speed of centrifugation should be set to decrease the content of adhered cells.

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