New research reveals the start of Huntington'sdisease elife
FCS 3.1 files resulting from assay were gated using an automated R-script running in flowCore. Prior to gating, the forward scatter , side scatter , donor fluorescence and autofluorescence channels were transformed using a logicle transform in R. Gating was then done using flowCore by sequentially gating for cells using FS00.A vs SS02.A then selecting for single cells using FS00.H vs FS00.W and finally selecting for expressing cells using FL03.A vs FL17.A.
Gating for cells was done using a rectangular gate with values of Xmin=2.7, Xmax=4.8, Ymin=2.7, Ymax=4.7). Gating for single cells was done using a rectangular gate with values of Xmin=4.45, Xmax=4.58, Ymin=2.5, Ymax=4.4. Gating of expressing cells was done using a polygon gate with x/y vertices of , , , . Cells falling within all of these gates were then exported as FCS3.0 files for further analysis.
The FCS files resulting from the autonomous gating in Step 1 were then utilized for the JAVA-based quantification of a curve similar to the analysis found in the original assayThe quantification procedure first divides a defined negative control DAmFRET histogram into 64 logarithmically spaced bins across a pre-determined range large enough to accommodate all potential data sets. Then upper gate values were determined for each bin as the 99percentile of the DamFRET distribution in that bin.
This gate is then applied to all DAmFRET plots to define cells containing proteins that are either positive or negative . In each of the 64 gates, the fraction of cells in the assembled population were plotted as a ratio to total cells in the gate.Cells were imaged using a CSU-W1 spinning disc Ti2 microscope and visualized through a 100x Plan Apochromat objective . mEos3.
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