Chapter 3 74 Framework Criteria What to report Please complete each criterion 6.1 Completion of MIFlowCyt checklist. Complete MIFlowCyt checklist criteria 1 to 4 using the MIFlowCyt guidelines. Template found at www.evflowcytometry.org. The MIFlowCyt checklist v1.0.0 has been completed and attached in the Supplementary Information. 6.2 Calibrated channel detection range If fluorescence or scatter calibration has been carried out, authors should state whether the upper and lower limits of a calibrated detection channel were calculated in standardized units. This can be done by converting the arbitrary unit scale to a calibrated scaled, as discussed in Section 4.3 and 4.4, and providing the highest unit on this scale and the lowest detectable unit above the unstained population. The lowest unit at which a population is deemed ‘positive’ can be determined a variety of ways, including reporting the 99th percentile measurement unit of the unstained population for fluorescence. The chosen method for determining at what unit an event was deemed positive should be clearly outlined. The lower fluorescence threshold for Ch01 (BV421), Ch02 (CFSE), and Ch05 (APC) was set at 110, 170, and 170 a.u., respectively. These values were obtained by analysing blanc-fluorescent Rainbow Calibration Particles (RCP-05-5, lot AL01, Spherotech), unstained PPP samples, and isotype control PPP samples. When scaled to ERF units, these values translated to 677.71 / 35.40 / 6.40 ERF, respectively. Upper fluorescent limits (high-end gating cut-off) for Ch01 (BV421), Ch02 (CFSE), and Ch05 (APC) was set at 100.000, 50.553, and 10.302 a.u., respectively. These gating cut-offs were determined to encompass all obtained fluorescent events. When scaled to ERF units, these values translated to 112,201 / 3776 / 123 ERF, respectively.
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