Necrosis HT-29

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 3 matching solutions for this experiment

FITC Annexin V Apoptosis Detection Kit I

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
• A total number of 10,000 cells per sample were analyzed using a BD FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA).	• Wash cells 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.	• Fluorescence to be 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 can be analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

Upstream tips
• A total number of 10,000 cells per sample were analyzed using a BD FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA).
Protocol tips
• Wash cells 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.
Downstream tips
• Fluorescence to be 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 can be analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

Manufacturer protocol Publication protocol 1 Relevant paper

Acridine Orange solution

Sigma-Aldrich

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
• Dual Staining Procedure for differentiating Live/ Apoptotic/Necrotic cells	• Seed 3x10^4 cells onto a 10mm coverslip and incubate overnight to form a confluent monolayer	• The operator to be blinded to the experimental groups and random fields to be selected (40X objective). • A total of 300 attached cells per cover-slip can be morphologically identified and counted as being either necrotic (red/orange nuclei),apoptotic (green condensed or fragmented nuclei) green or live (green non-condensed ovoid or rounded nuclei)

Upstream tips
• Dual Staining Procedure for differentiating Live/ Apoptotic/Necrotic cells
Protocol tips
• Seed 3x10^4 cells onto a 10mm coverslip and incubate overnight to form a confluent monolayer
Downstream tips
• The operator to be blinded to the experimental groups and random fields to be selected (40X objective). • A total of 300 attached cells per cover-slip can be morphologically identified and counted as being either necrotic (red/orange nuclei),apoptotic (green condensed or fragmented nuclei) green or live (green non-condensed ovoid or rounded nuclei)

Publication protocol 1 Relevant paper

Apoptosis/ Necrosis Assay Kit (blue, green, red) (ab176749)

Abcam

Upstream tips	Protocol tips	Downstream tips
• 2.5x10^4 cells/well were grown in 24-well plates		• Fluorescence of cells was examined and quantified.

Upstream tips
• 2.5x10^4 cells/well were grown in 24-well plates
Downstream tips
• Fluorescence of cells was examined and quantified.

Manufacturer protocol 1 Relevant paper

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