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The reason for using PI staining was that in addition to its ability to penetrate the nucleus in a dead cell, PI is also perfect for staining of cell walls, normally emitting red fluorescence.
In both types of invasion, T. cruzi induces host cell PI 3-kinase (PI3K) activity [2], [3].
During the formation of these bristles (around 12 hours After Pupal Formation, APF), N-mediated lateral inhibition is involved in selecting the primary precursor cell (pI) from a cluster of equivalent cells, called a proneural cluster.
Inside the cell, PI forms a fluorescent complex with nuclear DNA.
THI, EtHgCl, and TSA were titrated from 50 nM to 10 μM, IDC and MDC subsets were treated for 20 hr, and cell PI permeability was measured by flow cytometry.
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It remains a theoretical possibility, however, that a Plasmodium infection activates host cell PI-kinases in the red blood cell cytoplasm resulting in elevated PIP2 levels that would be inaccessible to the parasite's PI-PLC enzyme.
For transfected cells, PI fluorescence was measured in populations gated for AmCyan fluorescence.
Appropriate negative controls included unstained cells, PI only, secondary antibody only.
In order to determine late apoptotic cells, PI was added to the samples.
To discriminate between viable and dead cells PI (75 μM) (Sigma-Aldrich) was added to the mixtures before analyzes.
The percentage of the ratio of Ki-67 positive cells (PI, also expressed as a percentage) was calculated.
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