Necrosis HCT 116

A key signature for necrotic cells is the permeabilization of the plasma membrane. Necrosis can be quantified by several cellular and biochemical assays. When studied minutely, it reveals the difficulty in confirmation in secondary induction of necrosis in apoptotic cells. Apoptotic cells are being analyzed to shift to necrotic status owing to membrane permeability at later stages, and thus, discrimination of two cell death becomes critical. Therefore, it is crucial to use a necrosis detection kit or a defined procedure to analyze this unprogrammed form of death in response to immense chemical and physical insults.

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Found 4 matching solutions for this experiment

Upstream tips
• Assay measures the real-time exposure of phosphatidylserine (PS) on the outer leaflet of cell membranes during the apoptotic process.
• After cells were attached, 2× Sur-X of finally detected concentrations and 2× detection reagents in RealTime-Glo™Annexin V Apoptosis and Necrosis Assay (Promega,JA1011) were mixed in 100μl pre-warming culture medium and the mixture was added to each well.
Protocol tips
• The RealTime-Glo™ Annexin V Apoptosis and Necrosis assay is non-lytic and the simple “add-and-read” method allows multiple readings from a single assay well.
• The combination and timing of luminescent (annexin V binding) and fluorescent (DNA release) signals is used to differentiate secondary necrosis occurring during late apoptosis from necrosis caused by other cytotoxic events.
Downstream tips
• Relative luminescence units (RLU) and relative fluorescence units (RFU) were recorded over time up to 6 h by the spark multimode microplate reader (Tecan Trading AG, Switzerland).
Upstream tips
• Cells were washed with PBS, centrifuged and stained with Annexin V-FITC for 20 min at room temperature in the dark and subsequently stained with propidium iodide (PI) for 5 min before the measurement.
Protocol tips
• A total number of 10,000 cells per sample were analyzed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Fluorescence was detected via a 530/30 nm band-pass filter (FL-1; Annexin V—FITC) and a 670 nm long-pass filter (FL-3; PI). Raw files were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).
Downstream tips
• For the measurement of the viability and phosphatidylserine externalization as a marker of apoptosis, a BD Pharmingen FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) was used according to the manufacturer’s instructions.
Upstream tips
• Readily distinguishes between healthy, early apoptotic, late apoptotic and necrotic cells.
• Optimized for both fluorescence microscopy and flow cytometry applications.
• Suitable for death pathway analysis and drug/toxin studies.
Protocol tips
• Applications: Flow Cytometry, Fluorescence microscopy, Fluorescent detection

Acridine Orange solution + Ethidium bromide solution

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