Investigation of light cell interaction in optical microscopy
| Main Authors: | Krieger, Sigurd, Störberl, Anna S |
|---|---|
| Format: | info Proceeding |
| Bahasa: | eng |
| Terbitan: |
, 2019
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| Subjects: | |
| Online Access: |
https://zenodo.org/record/3547442 |
Daftar Isi:
- To test the potential light induced damage of cultured cells we developed a surface-mounted LED array containing 48 LEDs with selected 8 replicates of 6 different wavelengths (357, 465, 525, 588, 625 and 940 nm). A multistep encapsulation process was applied to prepare the electronic circuits for operation in state-of-the-art cell incubators. We employed sterile, cell culture treated, 96-well black microtiter plates containing a UV translucent clear bottom to culture and expose a standard cell line of human lung fibroblasts to different wavelengths for various time ranges.Generally, the production of cellular peroxides is known to lead to unwanted cell signaling side effects including apoptosis (programmed cell death). We used the cell permeable, peroxide activable, fluorescent dye (5,6-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate) CM-DCFH-DA to quantify the light induced peroxide formation. Light induced apoptosis is basically dependent on two major pathways, one direct genotoxic effect based on DNA damage by introducing DNA structures like pyrimidine (6-4) pyrimidone photoproducts and cyclobutane pyrimidine dimers (CPD), on the other side is singlet oxygen induced peroxide formation a potent inducer of cofilin dependent, caspase-2 independent apoptosis. To separate both pathway we planned to use singlet oxygen and peroxide scavenger, NaN3 and sodium pyruvate respectively, to suppress peroxide induced apoptosis and to measure the net effect on caspase-2 dependent DNA damage induced apoptosis. At all wavelengths the peroxide production was blocked by both components at 10mM concentration in CM-H2DCFDA loaded cells.This experiment demonstrated that NaN3 is an effective inhibitor of singlet oxygen production and can be used to block peroxide formation in human lung fibroblasts. In ongoing experiments, the apoptosis induction in the presence of NaN3 or an irreversible caspase-2 inhibitor Z-VDVAD-FMK is planned to separate the apoptosis inducing pathways.