Assessment of cytotoxicity or cell stress was analyzed by estimation of adenylate kinase (AK) [18 (link)] release from the endothelial cells (EPI-EC; HBMEC) and astrocytes (EPI-Astro; HA) subsequent to DAPK inhibition. Cells were seeded in multiple 12-well plates at a density of 1 × 105 cells per well and left overnight to adhere. Then DAPK inhibitor (100 μM) was prepared in the respective media and incubated for 24 h. Cell morphology of the brain endothelial and astrocytes were periodically monitored. Media samples were collected before and after the treatment with DAPK inhibitor. The detection was performed with the use of ToxiLight™ Non-destructive Cytotoxicity BioAssay Kit (Lonza, NJ, USA) following manufacturer instructions. The detection reagent was added to the samples, and photon emission indicating the presence of ATP was recorded by spec-trophotometer (BioTek, Synergy HT, USA).
Protocol detail
Evaluation of Cytotoxicity on Brain Cells
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Adenylate kinase
Astrocytes
Bioassay
Brain
Cells
Cytotoxicity
Endothelial
Endothelial cells
Inhibition
Corresponding Organization : Case Western Reserve University
Other organizations : Cleveland Clinic
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Variable analysis
independent variables
- DAPK inhibitor (100 μM)
- Adenylate kinase (AK) release from the endothelial cells (EPI-EC; HBMEC) and astrocytes (EPI-Astro; HA)
- Cell morphology of the brain endothelial and astrocytes
- Cell density (1 × 10^5 cells per well)
- Incubation time (24 h)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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