美天旎流式bmq01tfh111212-translation.pptVIP

* Here is a simple example how can gating can be used to determine the statistics of a plot. This is a typical Histogram. Shown here is a staining of human Peripheral Blood Mononuclear Cells (PBMC) with an APC-conjugated antibody against the surface molecule CD20. [Which cell type does express CD20?] The cell population on the left end of the scale has a low fluoresence intensity for CD20, they are CD20-; the population on the right end of the scale has a high fluorescence intensity. Besides the graphical display of this data also a statisitcal analysis can be doneby the use of gating tolls: To obtain statistical data for the two populations so called regions or markers (P5 and P4) can be defined. For the cell population included in such a region a table with statistical values can be created. There are two main readouts for flow-cytometric data: The frequency describes proportion of a cell population in per cent. The Median describes the average fluoresence intensity of the respective cell population. * Example for data analysis using a dot plot; two different parameters can be analyzed at once. To get the statisitcs for each population so called quadrants can be introduced. They are named after their location within the dot plot: LL = lower left UR = upper right etc…. For each population the frequency and average fluoresence intensity is displayed: CD3+CD4+ cells are in the upper right quadrant, their frequency within the gated cells is: 10.67%, CD3-CD4+ cells are in the upper left quadrant and their frequency is 40.33% The median fluorescence intensity for the CD4 staining is 41.36 for the CD3+CD4+ cells The median fluoresence intensity for the CD3-CD4+ cells is 4.16. That means that these cells have a ten-fold lower intensity for the CD4 staining than the CD3+CD4+ cells. * Why can dead cells cause analytical problems and how do these problems influence the results? Dead cells have the tendency to bind antibodies unspecifically. That means, even i