The Chicken Erythrocyte Nuclei (CEN) and Trout Erythrocyte Nuclei (TEN) standards I purchased from Biosure Corp. exhibit very nice DNA peaks with excellent CV's on my instrument. Each is also very consistent internally in DNA fluorescence. However, the two standards do not seem to compare with one another in my preparations. I would expect the trout (published ~5 pg DNA per 2C nucleus) to exhibit roughly twice the fluorescence as the chicken (published ~2.5 pg DNA per 2C nucleus)when both are stained and analyzed similarly. Regarding the comparison between the 2C CEN and TEN DNA content: the known 2C DNA content of chicken is published as 2.33 pg DNA per nucleus (it is also published as 2.5 pg). The published value for rainbow trout is published 2C = 5.17 pg DNA per nucleus. Thus the 2C TEN / CEN DNA ratio is 5.17 / 2.33 = 2.21 . However, when both CENs and TENs are DNA stained similarly and analyzed by flow cytometry, the mean peak value ratio for 2C TEN/CEN is nowhere near 2.21, at least in my preparations and on my instrument (Coulter EPICS ALTRA). The fluorescence peak ratio I measure is closer to 4.1 . Can anyone suggest why? I realize that different trout broodstocks and chicken lines may have slightly differing DNA contents, but I am not aware of strain-specific differences of this magnitude. Thus I am unable to satisfactorily resolve this issue. I am using a DNA fluorochrome (SYTOX Green) that does not exhibit base-pair specificity, and I have checked the linearity of the signal amplification system using fluorescent beads - it appears fine. In the excellent paper by Vindelov et al. in 1983 (Standardization of flow cytometric DNA analysis by use of chicken and trout rbc's as reference standards), the authors use both chicken and trout rbc's and "corrected the TRBC:CRBC ratio by changing the zero-point so that the ratio for each histogram was equal to 2.28 . Can anyone tell me how this is done (i.e what exactly is the parameter one adjusts to "change the zero point" such that a biologically correct DNA peak fluorescence ratio can be achieved between these two standards? I am relatively new to flow cytometry, and it is quite possible that I am simply missing some fundamental aspect in this standards comparison. Any advice or information anyone can offer is greatly appreciated.
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