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In retrospect our flow cytometry approach does not distinguish among different EC subtypes, however has proven very specific for only EC as the majority of sorted cells are positive for vWF (>99.9) and other endothelial markers.
We confirmed that the majority of sorted CD25+FR4+ CD4+ T cells express Foxp3 (Fig. 3A).
Following purification, fluorescence and DIC microscopy show that the majority of sorted neurons are TdTomato+ (images on right).
Similarly, while the majority of sorted MB and BPC lacked CK8 expression, there was a sub-population of EpCAMlo/neg/CD49f+ cells that co-expressed both CK8 and CK14 markers.
The purity of sorted Treg reached up to 95% determined by the expression of Foxp3 among sorted CD4+CD25+ T cells, indicating that the large majority of sorted cells belonged to the Treg lineage.
As shown in Fig. 3a, HMFs showed the highest enrichment for the stromal signature suggesting that the vast majority of sorted EpCAM−CD49f−/low cells from normal breast tissue are indeed fibroblasts.
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Typically one to five transformants were obtained per sorted droplet (using the high copy plasmid pASK-IBA63b-plus with ∼1000 plasmids per cell), thus ensuring that DNA from the majority of the sorted droplets was recovered.
Although we cannot rule out that our muscle EC FACS-sorted population may contains rare endothelial precursors, the EC population in our multicolor flow cytometry assay is defined as CD31 positive cells and the vast majority of the freshly sorted EC express vWF and eNOS.
Instead of sorted by popularity, they're sorted by activity (most recently active first).
Beside reference-free methods [ 22, 23], the majority of methods for whole blood are based on existing reference database of sorted blood cells by using differentially methylated loci across major leukocyte types [ 8, 14, 18].
To assess the purity of sorted cells, reanalysis of sorted cell populations was performed at various times during sorting.
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