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.

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 3 matching solutions for this experiment

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).

Acridine Orange solution + Ethidium bromide solution

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)
Upstream tips
• 2.5x10^4 cells/well were grown in 24-well plates
Downstream tips
• Fluorescence of cells was examined and quantified.
Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms