engleski

Apoptosis induced by dexamethasone and temperature stress on animal cells in monolayer or suspension

The death of a cell was thought for many years to be an uncontrolled, degenerative and catastrophic failure of homeostasis in response to cellular injury and was thus of little scientific interest. However, the discovery over the past two decades that all cells within multicellular organisms have the ability to activate a cell death programme termed apoptosis has challenged this idea and resulted in a re-evaluation in all aspects of biology including immunology, development, carcinogenesis, and more recently, toxicology. At the early stages of apoptosis, changes occur at the cell surface. One of these plasma membrane alterations in the translocation of phospatidylserine (PS) from the inner side of the plasma membrane to the outer layer. Using Annexin-V-fluorescein and propidium iodide (PI) for the differentiation from necrotic cells performed the analysis of phosphatidylserine on the outer leaflet of apoptotic cell membranes. Annexin V is the Ca^2+ dependent phospholipid-binding protein with high affinity for PS. In this work BHK 21 C 13 cells growing in suspension and MPK (minipig kidney) cells growing in monolayer were used as a model in detection of apoptosis. Cells were cultivated in Glasgow BHK 21 medium supplemented with 5% Calf bovine serum (CBS) in atmosphere of 5% CO_2, 95% air at 37°C. Monolayer of MPK cells have been developed homogeneous after 4 days on area of 9, 0 cm^2 at Chamber slide flasks. Initial concentration of MPK cells was 1, 5 x 10^5 cells/ml. Volume of media was 3 ml and cell viability was 70.1%. After the monolayer has been achieved, media was removed and 300 microL of 1mM dexamethasone solution in PBS was added on the monolayer and kept for 4, 5 hours at CO_2 incubator on 37°C. Dexamethasone solution was washed out from the monolayer using PBS, staining solution made of Annexin-V-fluorescein, propidium iodide and Hepes buffer (100 microL) has been added for 15 min at the 23°C, and the apoptosis was measured with the fluorescent microscope under filters range 450-490 nm and 510-560 nm. Apoptosis was also induced with high temperature (50°C). After the monolayer has been achieved Chamber slide flask with monolayer of MPK cells has been deposited at 50°C for 15 min. Essential medium was washed and staining solution made of Annexin-V-fluorescein, propidium iodide and Hepes buffer (100 microL) has been added for 15 min. Apoptosis was measured with the fluorescent microscope. BHK animal cell sample (in suspension) with viability of 57% after storage for 26 days at 4°C has been detected to determinate ratio of necrotic and apoptotic cells between 43% death cells. 3 ml suspension of BHK animal cells was centrifuged (800 rpm/ 2 min). Medium was removed and cells were washed with PBS and centrifuged again. 200 microL staining solution was added on cell pellet and incubated 15 min at the room temperature. Light and fluorescent microscope analyzed samples. Results showed that apoptotic cells could be differentiated from necrotic cells by the propidium iodide staining. Necrotic cells take up propidium iodide and staining orange/green while apoptotic cells stain green only.

apoptosis; BHK 21 C13 cell line; flow cytometry; phosphatidylserine (PS); Annexin V

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