(A) Multiparameter fluorescent beads are analyzed with logarithmic acquisition and linear data array.
Histograms C and D show the effect of stimulation with the tumor cell lysate on the same cells. Histograms A and B show the background expression of CD69 on NK and T cells, respectively, incubated in the absence of the tumor cell lysate.
FLOWJO 10 NORMALIZATION HISTOGRAM PLUS
An example is presented below:Ī bead preparation containing unlabeled beads plus seven bead populations expressing increasing amounts of fluorochrome was analyzed by flow cyto-metry, and the resultant fluorescent peaks were arrayed as log-amplified data (Fig. These values are only semiquantitative but can be used to standardize experiments between runs or between cytometers. These beads each have a nominal value of fluorescence intensity, termed molecules of soluble fluorochrome/bead, ascribed to them so that individual median channel values can be converted into a standardized numerical value. A huge variety of latex beads are available commercially, but for fluorescent semiquantitation purposes, a kit containing a minimum of five populations of beads with increasing fluorescence intensities should be used. For this, there are fluorescent standards and these are most commonly and reproducibly provided by latex beads.
The measurement of relative fluorescence intensity in this case is quite adequate for the required purpose, but to compare experiments run on different days or between collaborating centers, it is valuable to have some form of semiquantitative measurement. Plainly, the NK cells have shown considerably greater activation in response to the tumor cell lysate than the T cells and the relative fluorescence intensities of the two populations can be compared with each other or with their nonstim-ulated state. A relative fluorescence intensity may be calculated by expressing the increased CD69 expression as a percentage of the level of expression on the T cells as follows:
It is not possible to conclude that the CD69 expression is more dense on the NK cells, because there is no certain measurement of relative cell size all that can be determined is that there are more CD69 molecules on the NK cells than their matched T cells. NK cells show a higher proportion of CD69+ cells than do the matched T-cell population, and the intensity of expression of CD69 is higher on the NK cells as measured by the median channel fluorescence intensity (MedCF). Figure 4 histograms A and B show the background expression of CD69 on NK and T cells, respectively, incubated in the absence of the tumor cell lysate. Mononuclear cells were incubated overnight in the presence or absence of a tumor cell lysate and labeled with anti-CD3, anti-CD69, anti-CD16, and anti-CD56. Taking a single median value from a fluorescence signal that contains multiple log-normal distributions should be avoided (e.g., the PI signal in Figure 3A, which shows at least three subpopulations).Ī typical experiment in which one might want to measure relative fluorescence intensity is shown here, where the effect of tumor cells on the expression of CD69 on resting NK cells and T cells from the same donor was investigated. Median fluorescence intensity is a value that should be taken from a single log-normal distribution. The best estimate of the average of log-arrayed fluorescent signals is the median or geometric mean, so always assess " median fluorescence intensity" rather than mean. This means that the distributions, although appearing Gaussian, are actually "log normal" distributions in which the mean fluorescence intensity will be skewed to the right that is, the mean will overestimate the true average fluorescence intensity. In most cases, when fluorescent signals derived from mAb binding are measured, the data are log-transformed to provide sufficient resolution of the cells. The only difficulty with such a comparison is determining the average level of fluorescence. In most cases, the measurement of "relative fluorescence intensity" is adequate, where the fluorescent channel number that best approximates the average fluorescence of one population is compared with the same value from a second population labeled with the same reagent. These measurements may be relative, semiquantitative, or quantitative depending upon the question asked and the reagents available. A regular use of flow cytometers is the determination of the density of specific molecules on the surface of one or more cells in a population.
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