Wanted dead or alive – Tips for investigating cell death (Part 1) – autophagy and apoptosis

Cell death research has become an intensive research topic as disturbance in the natural cell death process is contributing to several different diseases ranging from neurodegenerative diseases to AIDS, cancer and autoimmune diseases. BioNordika provides products for both inducing and detecting different cell death modes. Keep reading to get some tips for your research! (1) The first part of the blog is about autophagy and apoptosis. The second part will describe the inflammatory forms of cell death.

Autophagy

Autophagy is not actually a form of cell death, but rather a process to prolong the life of cell in stress conditions. In this process, the cell breaks down damaged or abnormal proteins, organelles in cytoplasm. The Nobel prize in Physiology or Medicine was given to Dr. Ohsumi on discoveries of mechanisms for autophagy. Autophagy and Apoptosis are in constant cross-talk with each other and the fine balance between these processes is interesting in pathologies like cancer and infection in tissue, but it is also part of normal development. Excessive autophagy can lead to cell death.

Activation: Rapamycin (mTORC1) (13346) Cayman, nutrient deprivation

Tips for detecting autophagy:

The most important signaling pathway for autophagy is the mTOR kinase pathway. Activated mTOR (Akt and MAPK signaling) suppresses autophagy, whereas negative regulation of mTOR (AMPK and p53 signaling) promotes autophagy. The full signaling pathway can be downloaded from the link below. In an ongoing autophagy, Atg12 and Atg5 are covalently bound and targeted to autophagosomic vesicles.

Autophagy Antibody Sampler Kit #4445 (Cell Signaling Technologies) (contains enough antibody to perform two Western Blot experiments for following target antigens:

Beclin-1, LC3A/B, Atg5, Atg12, Atg16L1, Atg7, Atg3

Apoptosis

Apoptosis is a highly controlled form of cell death, which does not alert immune system. Apoptosis can be recognized from bleb formation. These vesicles are called the apoptotic bodies. Apoptosis is mediated by a family of protein called caspases. Apoptosis cascades are differentiated to intrinsic and extrinsic pathways. Intrinsic is initiated by cytochrome C release from mitochondria whereas extrinsic pathway is activated by external ligands binding to cell death receptors. (3)

Activation: DNA damage, Actinomycin D (#11421 Cayman chemicals), Obatoclax (mesylate) (#11499 Cayman chemicals)

Tips for detecting apoptosis:

• Aquaphile JC-1 #70076 (Biotium). In healthy cells, the dye localizes to mitochondria with red fluorescence, while in apoptotic cells with depolarized mitochondrial potential, the dye localizes in the cytoplasm and fluorescent green.
• Annexin V-FITC early apoptosis detection kit (flow cytometry) #6592 (Cell Signaling Technology)
• TUNEL kits (IF, F) different fluorophores (#25879 488 nm, #64936 640 nm and #48513 594 nm)
• Cleavage of Caspase-3 (Western Blot)
  • Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb #9664
  • Caspase-3 Antibody #9662
  • Caspase-3 Control Cell Extracts #9663

For more tips and available cell death products, get in contact with our product specialists at info@bionordika.se

Next blog – Wanted dead or alive – Tips for investigating cell death (part 2) – Necrosis, necroptosis, pyroptosis

References:

• https://ki.se/en/research/facts-about-cell-death
• Codogno P, Mehrpour M, Proikas-Cezanne T. Canonical and non-canonical autophagy: variations on a common theme of self-eating? Nat Rev Mol Cell Biol. 2011 Dec 14;13(1):7-12. doi: 10.1038/nrm3249. PMID: 22166994.
• Bertheloot D, Latz E, Franklin BS. Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cell Mol Immunol. 2021 May;18(5):1106-1121. doi: 10.1038/s41423-020-00630-3. Epub 2021 Mar 30. PMID: 33785842; PMCID: PMC8008022.

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