What does PI staining do?
Propidium iodide (PI) is a cell-impermeant DNA binding dye that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with a stoichiometry of one dye per 4-5 base pairs of DNA. Little or no sequence preference is observed.
What is PI uptake assay?
PI uptake is a live/dead assay and does not distinguish between apoptotic or necrotic cell death. However, since in most cell types shrinkage is observed in apoptosis but not in necrosis, a combination of reduced forward scatter properties and PI uptake can be used to estimate the mode of cell death.
Which of the following dye is most commonly used for cell cycle analysis using FACS?
Flow cytometry cell cycle analysis using propidium iodide DNA staining.
How does FlowJo analyze cell cycle?
FlowJo provides a simple interface to performing fairly sophisticated DNA/Cell Cycle analysis. To launch the Cell Cycle platform, select any sample or gated population (i.e., where you have gated out debris or gated for a desired phenotype), and choose “Cell Cycle…” from the “Workspace” menu.
Can PI stain live cells?
Generally live cells cannot be stained by PI.
Is PI cell permeable?
Both DAPI and Hoechst are living cell-permeable DNA stains, while PI is impermeable to cells with an intact plasma membrane and thus it can be used to distinguish between dead cells and living cells.
How do you identify cells in flow cytometry?
Generally, a specific cell type is marked with fluorescent dye (markers) like fluorophore or propidium iodide. The ability to use multiple fluorescent markers simultaneously allows for the identification of multiple cell types, as well as functional markers that further characterize each sample.
Why is the pulse width analysis important in cell cycle analysis in flow cytometry?
Fluorescence pulse width can provide size information on the fluorescence-emitting particle, such as the nuclei of propidium iodide-stained cells.
Is PI toxic to cells?
However, PI is toxic to cells – how long can a cell last with (a certain concentration of?) PI, before it degrades and can no longer be imaged? Thank you in advance for your help. Join ResearchGate to ask questions, get input, and advance your work.
What is PI positive cells?
Propidium iodide (PI) is a popular red-fluorescent nuclear and chromosome counterstain. Since propidium iodide is not permeant to live cells, it is also commonly used to detect dead cells in a population. PI binds to DNA by intercalating between the bases with little or no sequence preference.
How to prepare DNA for flow cytometry with PI staining?
For easy setup, with PI staining of DNA content for flow cytometry we recommend our Propidium Iodide Flow Cytometry Kit, otherwise, we recommend this protocol. Harvest the cells in the appropriate manner and wash in PBS. Fix in cold 70% ethanol.
How can I check viability of cells before staining with Pi?
They are also not appropriate to check viability, since the samples are fixed in EtOH prior to staining with PI.** Step 1: Harvest and count cells. Step 2: Centrifuge and remove supernatant. Vigorously vortex the pellet for 10 seconds and continue to vortex the cells while slowly adding 1 ml of ice cold 70% ethanol drop by drop to the pellet.
How can I measure the Pi signal of a cytometer?
This can be achieved either by using pulse area vs. pulse width or pulse area vs. pulse height depending on the type of cytometer. PI has a maximum emission of 605 nm so can be measured with a suitable bandpass filter. Forward scatter vs. PI signal; PI histogram.
How to analyze cell cycle status using flow cytometry?
In this unit, we describe two protocols for analyzing cell cycle status using flow cytometry. The first is based on the simultaneous analysis of proliferation specific marker (Ki-67) and cellular DNA content, which discriminates resting/quiescent cell populations (G0 cell) and quantifies cell cycle distribution (G1, S or G2/M, respectively).