Additional file 3.

Cytometry data preprocessing for figures 2and 4. Cytometry data for RPE1, stained for PCNA (FITC), Mcm-6 (PE), phospho-S10-histone H3 (A647), and DNA (DAPI) are shown. (A) aggregate and debris discrimination: singlet cells were included in region R1 based on integrated (UV-440-A) versus peak (UV-440-H) DAPI signal. All subsequent data were gated on R1. (B) Mitotic discrimination: mitotic cells were included in region R2 based on elevated histone H3 phosphorylation. All subsequent data were Boolean "NOT" gated on R2. (C) G2 discrimination: G2 and 4C G1 cells were included in region R3 based on 4C DNA content and absence of bound PCNA expression. All subsequent data except (D) were NOT gated on R3. (D) 4C G1 cell discrimination: 4C G1 cells (Mcm6 positive) and negative 4C cells were included in region R4. All subsequent data were NOT gated for R4. This is ~redundant with R3. (E) abnormal large and small cell discrimination: abnormally small events were included in R5. Large cells with low Mcm6 levels were identified as abnormal large G1 cells (i.e., based on size, they should have been Mcm6-high). Subsequent data were NOT gated for R5 and R6. (F) resultant plot after sequential Boolean logic (R1 NOT (R2 OR R3 OR R4 OR R5 OR R6)) applied to data. G1 (orange and cyan) and S phase (red-brown) data result. Data were also compensated conventionally for spectral overlap between FITC and PE (not shown). Removal of cells in R6 is conservative in a cell cycle sense. Their size suggests that they should have entered S phase, since they are larger than the average cell in early S. We see these in variable numbers in all three hTert immortalized lines with which we have worked. Since they express Mcm6 very low, they may be out-of-cycle for unknown reasons. If these cells are included, the information in Figures 2 and 4 - 6 do not change, suggesting that they represent an offset in the G1a compartment.

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Frisa and Jacobberger BMC Cell Biology 2010 11:26   doi:10.1186/1471-2121-11-26