How can you detect early and late apoptotic cells by flow cytometry?
As can be seen in Figure 2 below, live cells do not stain with either FLICA or a viability dye. An apoptotic cell will be positive for FLICA and fluoresce at a specific wavelength depending on fluorophore, whilst excluding the viability dye. Cells that are dead will be positive for both FLICA and the viability dye.
How does propidium iodide measure the cell cycle?
The most straightforward method for cell cycle analysis is to fix the cells with ethanol, treat with RNase, and stain with PI. Propidium Iodide stains RNA in addition to DNA, so cells must be treated with RNase to analyze cell cycle accurately. RNase treatment is not required when using DAPI.
What is flow cytometry technique?
Flow Cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument.
How do you measure apoptosis?
Apoptosis is detected by measuring the externalization of phosphatidylserine on the plasma membrane using fluorescent-tagged annexin V. Additionally, flow cytometry can be employed to determine alterations in cell size (Bortner and Cidlowski, 2001; Warnes et al.
What are early apoptotic cells?
Early apoptotic cells are Annexin V-positive and PI-negative (Annexin V-FITC+/PI−), whereas late (end-stage) apoptotic cells are Annexin V/PI-double-positive (Annexin V-FITC+/PI+)3. Other staining approaches use fluorescence-conjugated antibodies, which specifically bind to intracellular apoptotic markers.
How does propidium iodide work in flow cytometry?
Flow Cytometry Cell Viability Overview Flow cytometry is a quick and reliable method to quantify viable cells. Propidium iodide (PI) is a membrane impermeant dye that is generally excluded from viable cells. It binds to double stranded DNA by intercalating between base pairs.
How does propidium iodide staining work?
Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. PI also binds to RNA, necessitating treatment with nucleases to distinguish between RNA and DNA staining.
Is flow cytometry expensive?
Although flow cytometry is the “gold standard” for accurate and automated measurement of CD4 T lymphocytes, the technique is expensive and requires sophisticated equipment as well as trained personnel to perform it.
What is FSC and SSC in flow cytometry?
In flow cytometry, the light scattered by cells is measured by two optical detectors: forward scatter (FSC) that detects scatter along the path of the laser, and side scatter (SSC) which measures scatter at a ninety-degree angle relative to the laser.
What are apoptosis assays?
An apoptosis assay detects and quantifies the cellular events associated with programmed cell death, including caspase activation, cell surface exposure of phosphatidylserine (PS) and DNA fragmentation.
What is the purpose of the Nicoletti assay?
The Nicoletti assay, named after its inventor, the Italian physician Ildo Nicoletti, is a modified form of cell cycle analysis. It is used to detect and quantify apoptosis, a form of programmed cell death, by analysing cells with a DNA content less than 2n (“sub-G 0 /G 1 cells”).
What kind of buffer is used in the Nicoletti method?
The Nicoletti method is very similar to that described above, with the exception that a hypotonic buffer (such as HFS buffer containing sodium citrate and Triton X-100, or a hypotonic fluorochrome solution) is used to permeabilize the cells.
Why is assay technology important to the workplace?
Assay Technology desires to maintain its long-held position as technology leader for personal monitoring of chemicals in the workplace.