This work highlights the potential signaling and phenotypic diversity of GSCs. GSCs that highly expressed all 4 surface markers had the greatest self-renewal capacity, WNT inhibitor sensitivity, and in vivo tumorigenicity. Once in culture, some subpopulations were lost and previously undetectable ones materialized. Using mass cytometry and fresh operating room specimens, we found 15 distinct GSC subpopulations in patients, and they differed in their MEK/ERK, WNT, and AKT pathway activation status. It remains unknown how these single-surface marker–defined GSC populations compare with each other in terms of signaling and function and whether expression of different combinations of these markers is associated with different functional capacity. Since their discovery in 2003, GSCs have been isolated using single-surface markers, such as CD15, CD44, CD133, and α 6 integrin. A major therapeutic goal is to target GBM stem cells (GSCs), a subpopulation of cells that contribute to treatment resistance and recurrence. Most patients with glioblastoma (GBM) die within 2 years. Chheda, 660 South Euclid, Campus Box 8069, St. Louis, Missouri, USA.Īddress correspondence to: Milan G. Louis, Missouri, USA.ħDepartment of Neurosurgery, Memorial Sloan Kettering Cancer Center, New York, New York, USA.ĨBrain Tumor Research Center, Massachusetts General Hospital, Boston, Massachusetts, USA.ĩDepartment of Neurology, Washington University School of Medicine, St. Louis, Missouri, USA.ĦSiteman Cancer Center, Washington University in St. Louis, Missouri, USA.ĥDivision of Biostatistics, Washington University School of Medicine, St. Louis, Missouri, USA.ĤBiomedical Engineering and Center for Biological Systems Engineering, Washington University in St. This is typically done for antibody titrations and a titration curve is used to determine which signal/noise ratio is optimal for the antigen.Ĭonclusion: The experts recommend using the median (preferred) or the geometric mean (second best choice) for the evaluation of MFI on a logarithmic scale.2Center for Human Immunology and Immunotherapy Programs, andģDepartment of Neurosurgery, Washington University School of Medicine, St. Looking at the example B, this would result in a S/N ratio of 77:1 for the CD7. If compensation settings are applied, the MFI's of the antigen-negative as well as the antigen-positive populations are often affected. A more meaningful way may be to measure the signal to noise ratio (mean of an antigen-positive population / mean of antigen negative population) while no compensation setting are applied. voltage, compensation, antibody dilution, tandem dye degradation, laser fluctuations, etc.), it can be misleading when comparing intensity of any kind across multiple experiments. Fluorescent intensity is sensitive to experimental condition (e.g. Gating each population and presenting percentages will provide much more useful information.Īlthough the MFI is often used to define and describe the mean intensity and level of antibody expression, it should be noted that this assumes that the instrument is optimized including the voltage and compensation settings. The MFI should NOT be used in a bimodal distribution (example C) as any average only holds true for normal distributions, and a bi-modal population is by definition not normal. In order to compensate for this, the geometric mean (gMFI) is often used to account for the log-normal behavior of flow data. However, if we look at example B, a skewed antigen expression causes the mean to drift in the direction of the skewed area (in this case to the left). In an even distribution (example A) the median, arithmetic mean, mode and the geometric mean is almost identical. Mode: refers to the channels which are most frequented. Geometric mean: Without going into too much mathematical detail, the geometric mean compensates for that and is considered the second best choice of describing the MFI of a logarithmic histogram.Ĥ. Because fluorescent intensity increases logarithmically (and most flow data are logarithmic), arithmetic mean quickly becomes useless to generalize a population of events, as a right-hand skew causes even more exaggeration of the mean and accurate MFI measurements cannot be made of events off scale either at the negative/dim or bright ends of the histogram.ģ. Preferred method to measure MFI of a logarithmic histogram.Ģ.Ěrithmetic mean: number of events in each fluorescent channel divided by the number of channels. Median: midpoint of population (middle channel). However, it is important to know which kind of mean we are talking about.ġ. MFI is typically understood as mean fluorescence intensity.
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