Principles of Flow Cytometry文档.ppt

* * * * * * * * * * * * * * * * * * * * * * * * * 1 * 2 * * 1 * 7 * 8 * 9 * 12 * 13 * 15 * 1 * * * * * * * * * * * * * * * * * * * * Compensation Controls  Single Stain Controls * Does not matter as long as: The autofluorescence is the same in the negative and positive populations you are lining up. eg, Pre-gate on lymphocytes if you are using CD8 FITC as a single stain control The compensation values will be valid for ALL cell types, regardless of which type of cell is used to calculate the values. The compensation is specific for the fluorochrome, not the cell type Single Stain Controls - Which cells? * Use the same reagent (Ab-fluorochrome conjugate) as used in the experimental sample… OR A different antibody may be substituted, as long as it is conjugated to the same fluorochrome. However… Single Stain Controls - which reagents? * Caveats for substituting reagents: Controls should be as bright as possible As bright or brighter than the experimental stains GFP, CFSE, and FITC are NOT the same fluorochrome even though they are all green! With tandem dyes (Cy5PE/Cy7PE etc.) it is necessary to use the exact same reagent spillover varies from reagent to reagent  Single Stain Controls - which reagents? * Compensation of tandem-conjugates can differ from lot to lot * Use same reagent as experimental sample Lots positive Small CV, bright Some reagents won’t work (IgL, EMA/PI) can mix with regular comps Using Antibody Capture Beads as single stained controls * Software Compensation  Automated Tools for Setting Compensation * Compensation Tools Must have single stained controls Software calculated compensation for you! Easy, accurate and quick. Makes MULTI- Color compensation possible * Software Compensation Tools Available on new generation machines DakoCytomation’s Summit (version 4) Coulter FC500 BD Diva Others Post-acquisition software FCS Express FCS Press WinList FlowJo Others * Compensation Matrix FL1 FL2 FL3 FL1 Comp 3.96 0 FL2 C