treatment of thp-1 cells with lps/atp for deciphering cell death mechanisms

Analyzing LPS/ATP-Triggered Cell Death Mechanisms in THP-1 Cells

Cell death is a fundamental physiological process in all living organisms. In recent years, substantial studies have shown that cell death is involved in the immunity and balance within the organism. Studying cell death helps us better understand the onset and development of diseases. Several forms of programmed cell death have been described, and some key targets in these processes have been identified. Pyroptosis, apoptosis, and necroptosis are the three genetically defined programmed cell death pathways involved in internal balance and disease1.
Pyroptosis is characterized by the formation of membrane pores and the release of cell contents. Activated caspases or granzymes cleave gasdermins to separate their N-terminal domains, which then oligomerize, bind to the membrane, and form pores2,3,4. The gasdermin pore provides an atypical secretion channel across cellular membranes, resulting in downstream cell responses, including content release and ion influx2,3,4. Ultimately, the cells eventually experience plasma membrane rupture and pyroptotic lysis facilitated by ninjurin-15. In apoptosis, activated Bax and Bak form oligomers on the mitochondrial outer membrane and release cytochrome C, which is regulated by a balance between proapoptotic and antiapoptotic proteins of the BCL-2 family, initiator caspases (caspase-8, -9 and -10) and effector caspases (caspase-3, -6 and -7)1,6,7. The morphological changes of apoptosis include membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and apoptotic body formation6,8. The receptor-interacting serine/threonine-protein kinase 1 (RIPK3) and mixed-lineage kinase domain-like (MLKL) are two downstream core components of the necroptotic mechanism1. RIPK3 recruits and phosphorylates MLKL, p-MLKL oligomerizes, associates with the cell membrane, initiates membrane perforations, causes ion influx, increases intracellular osmolarity, and eventually cell rupture6,9. Gasdermins and MLKL bind to the plasma membrane and mediate pyroptosis and necroptosis, respectively, while BAX/BAK mediates apoptosis by binding to the outer membrane of mitochondria6.
Although each pathway has specific mechanisms and outcomes, they lead to similar changes on the cell membrane. Under normal circumstances, phosphatidylserine (PS) is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, PS will be exposed, outside of the plasma membrane. Caspase-3/caspase-7 activates TMEM16 and XKR families, which leads to an asymmetrical cell membrane and externalizes&#160;PS during apoptosis10. Gasdermin D-mediated and MLKL-mediated Ca2+ influx leads to loss of the symmetry of the phospholipid bilayer on the cell membrane and the exposure of PS. The exposure occurs before the loss of cell membrane integrity11,12. Based on the similar changes in the membrane of these three types of programmed cell death, we use flow cytometry to analyze Annexin V/7-Aminoactinomycin D (7-AAD) double staining to detect cell death. Annexin V, a calcium-dependent phospholipid-binding protein, has a high affinity for PS, which can serve as a sensitive probe to detect exposed PS on the surface of the plasma membrane13. 7-AAD is a nucleic acid stain that cannot pass through the entire cell membrane. It is similar to propidium iodide (PI), a commonly used nucleic acid dye. They have similar fluorescence characteristics, but 7-AAD has a narrower emission spectrum and less interference with other detection channels. Owing to these similarities, it is not sufficient to distinguish between pyroptosis, apoptosis, and necroptosis. We used flow cytometry to detect the cell death from whole cells. A second method is used to capture the cell membrane using scanning electron microscopy (SEM) to observe the possible forms of cell death in individual cells.
We established a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. This protocol focuses on observing cell death rather than investigating mechanisms.

Author Spotlight: THP-1 Macrophage Response to LPS/ATP &amp;#8212; Unveiling the Pyroptosis, Apoptosis, and Necroptosis Spectrum

LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation

We are addressing LPS and ATP-introduced deaths of PMA-differentiated THP-1 macrophages. LPS and ATP-introduced deaths are not limited to pryoptosis but apoptosis and necroptosis can also occur. This will aid in a better understanding of cell death after LPS and ATP stimulation, and choosing more suitable experimental methods.Cryo-EM technology many studies the other structures in tissues, cells, and microorganisms. Cryo-EM provides neo native structure data for diverse molecular complexes. For example, using Cryo-Em techniques to perform 3D reconstruction can help us further understand the characteristic of apoptotic and necroptotic parts.Although pryoptosis is considered dominate in LPS and ATP-introduced cell death, one cannot determine the appropriation of these death forms or whether they all exist. Validating diverse cell death and measuring them will be our area of investigation in the future.

Treatment of THP-1 Cells with LPS/ATP for Deciphering Cell Death Mechanisms

treatment-thp-1-cells-with-lpsatp-for-deciphering-cell-death

Research

JoVE Journal

Biology

71 Görüntüleme.  Chengdu University of Traditional Chinese Medicine. Başlamak için, farklılaşmış THP-1 hücreleri elde edin ve harcanan ortamı kültür plakasının kuyularından çıkarın Hücreleri PBS ile yıkadıktan sonra, hücrelere lipopolisakkarit veya LPS içeren serum içermeyen ortam ekleyin ve plakayı inkübe edin. Daha sonra model grubuna adenozin trifosfat veya ATP stok çözeltisini ekleyin ve plakayı %5 karbondioksit ile 37 santigrat derecede 45 dakika inkübe edin. Daha sonra, tüm süpernatanı kültür plakasının kuyularından as...