Supplementary MaterialsAPPENDIX. in parallel to Flow-FISH. Telomere fluorescence of G0/1 cells of subpopulations and internal standards from Flow-FISH are normalized for DNA ploidy, and telomere size in subsets of interest is expressed like a portion of the internal standard telomere size. (Stewart and Stewart, 1997b)]. (Hoffman, 2005). 11. Obtain data using the following parameters: Collect cell-surface fluorescence and telomere fluorescence with log amplification, and DNA fluorescence with linear amplification using area and width signals for doublet discrimination. versus DNA content (Vehicle Ziffle et al., 2003). A DNA dye that provides more exact DNA distributions such as DAPI (Harley et al., 1990) is required for improved cell cycle determination. Ideally, samples should be acquired immediately after the completion of DNA staining, although Kapoor et al. (2009) found that no significant variations in the measurements were observed up to 24 hr. Assay Standardization Settings to account for inter-individual telomere size variability In order to right for the variability in telomere size between individuals of the same age (observe section on Factors Affecting Telomere Size for details), studies that investigated the telomere size in leukemic cells by Flow-FISH have used CD3 T cells from your same individual (which can be expected to become unaffected by the disease process) as an internal standard (Brummendorf et al., 2000; Drummond et al., 2004). Similarly, to better describe disease specific changes in telomere biology in immune cell subsets in Lupus individuals, Beier et al. (2007) utilized CD14+ monocytes like a subject-specific internal control. Strenuous statistical approaches such as multiple linear regression and appropriate coordinating of experimental organizations can be used to reduce the influence of clinical guidelines such as age, disease duration, grade of lymphopenia, and treatment within the Alfuzosin HCl telomere size assessment (Beier et al., 2007). Settings to account for experimental variability in Alfuzosin HCl dedication of telomere size Flow-FISH of telomere size depends on the reliability of the generation and measurement of a fluorescent signal for which minor alterations or erratic or systematic errors in the procedure can lead to relatively large changes in the readout. Therefore, for improvement of the accuracy of the assay, addition of a stable internal standard that settings for the variations between individual reaction tubes is critical, as it limits statistical errors from tube-to-tube and day-to-day analysis. Hultdin Alfuzosin HCl et al. (1998) have introduced the use of the 1301 cell collection, a subline of CCRF-CEM, as an internal standard. 1301 cells have extremely long telomeres ((Danzynkiewicz and Juan, 1997). For any ready assessment of data acquired in different laboratories, however, it is necessary to generate a linear regression collection between the Flow-FISH technique and the TRF fragment size in kilo bases as measured by Southern blotting (Hultdin et al., 1998; Law and Lau, 2001; Schmid et al., 2002). This can be achieved by parallel analysis of samples of numerous telomere Alfuzosin HCl lengths, e.g., human being samples from individuals that differ in age or cell lines that have long telomeres. After creating the correlation between TRF ideals and Flow-FISH data, the correlation equation can then be applied to subsequent samples that are processed with the same Flow-FISH method and analyzed on the same flow cytometer. Reporting of relative telomere length only allows the comparison of the results of experiments carried out with the same standard; however, it TRAF7 is also possible to express telomere lengths in absolute units i.e., base pairs. Rufer et al. (1998) reported their data in terms of arbitrary fluorescence units or Molecules of Equivalents of Soluble fluorochrome (MESF) units using as the Quantum MESF beads from Bangs Laboratories Inc. (Rufer et al., 1999). The FITC-labeled beads contain five different populations, each labeled with a known number of FITC molecules. The mean fluorescence intensity (MFI) value for each bead peak corresponds to the approximate number of fluorescein molecules; thus, a standard curve for MESF values, and by extension FITC molar concentration, can be generated. By doing side-by-side Southern blotting and Flow-FISH using a PNA probe conjugated with a known molar amount of FITC and.
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