fluorogenic caspase 3 substrate ac-devd-amc 14986  (Cayman Chemical)

Journal: Biomolecules

Article Title: Protective Effects of Cannabidiol (CBD) against Qxidative Stress, but Not Excitotoxic-Related Neuronal Cell Damage—An In Vitro Study

doi: 10.3390/biom14050564

Figure Lengend Snippet: The effects of cannabidiol (CBD) on the H 2 O 2 -evoked changes in reactive oxygen species level (ROS, ( A )), mitochondrial membrane potential (MMP, ( B )), caspase-3 activity ( C ), and cytotoxicity ( D ) in primary neuronal cell cultures. ( A ) ROS production was assayed with a CM-H 2 DFFDA probe, as described in details found in the Material and Methods section. ( B ) MMP measurement was performed after 6 h of treatment of cells with CBD (0.01–5 μM), NAC (1 mM), and H 2 O 2 (0.2 mM) employing a TMRE fluorescence probe. ( C , D ) Caspase-3 activity and cytotoxicity measurements were performed in cells treated for 9 h with CBD (0.01–5 μM) and H 2 O 2 (0.2 mM). A caspase-3 inhibitor, Ac-DEVD-CHO (Ac, 20 μM), was used as a positive control to the assay. After treatment, in cell lysates, caspase-3 activity ( C ) was measured using the fluorogenic substrate Ac-DEVD-AMC, and cytotoxicity was assessed in the cell culture medium (LDH test, ( D )). Data after normalization to vehicle-treated cells (100%) are presented as a mean ± SEM. * p < 0.05 and *** p < 0.001 vs. vehicle-treated cells; # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. H 2 O 2 - treated cells.

Article Snippet: Caspase-3 (Ac-DEVD-AMC) fluorogenic substrate was from Enzo Life Sciences (New York, NY, USA).

Techniques: Membrane, Activity Assay, Fluorescence, Positive Control, Cell Culture

Journal: Ecotoxicology and environmental safety

Article Title: A novel chemo-phenotypic method identifies mixtures of salpn, vitamin D3, and pesticides involved in the development of colorectal and pancreatic cancer

doi: 10.1016/j.ecoenv.2022.113330

Figure Lengend Snippet: (A) Representative images of HaCaT human skin keratinocytes incubated with indicated concentrations of salpn for 72 h in 96-well plates. (B) Dose-response for HaCaT skin keratinocytes cells after treatment with salpn showing a marked decrease in cell viability after exposure to salpn. The IC 50 (C) for salpn is 84 μM. Annexin/PI Apoptotic Assay of HaCaT cells treated by salpn for 72 h (C & D). (C) Representative images of HaCaT human skin keratinocytes in 6-well plates showing effects of exposure to increasing concentrations of salpn for 72 h. (D) Annexin-PI staining apoptosis data after treatment with salpn showing % of live cells, early, late and total apoptosis, respectively. Caspase-3 Activity Assay of HaCaT Cells treated with increasing concentrations of salpn for 72 h (E) . Induction of caspase-3 activity in Jurkat positive control cells exposed to etoposide (upper panel) and cells treated with different concentrations of salpn (lower panel). Zebrafish morphological assay (F) . Dose-dependent response toxicity of salpn measured as the lowest dose leading to significant developmental abnormalities at 2 days after post fertilization (2 dpf). TUNEL TMR apoptosis assay (G) with salpn treated embryos, dark dim background staining has either no staining (Negative control) or very few apoptotic cells (DMSO control). Arrows on the picture show the positive cells. Apoptotic cells in 2 dpf embryos were labeled using the in-situ Cell Death Detection Kit-TMR.

Article Snippet: Caspase-3 assays were performed to measure free 7-amino-4-methylcoumarin (AMC) using 100 μl reactions containing caspase-3 fluorogenic substrate Ac-DEVD-AMC (Invitrogen).

Techniques: Incubation, Staining, Caspase-3 Activity Assay, Activity Assay, Positive Control, TUNEL Assay, Apoptosis Assay, Negative Control, Labeling, In Situ

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Liver Cholesterol Overload Aggravates Obstructive Cholestasis by Inducing Oxidative Stress and Premature Death in Mice

doi: 10.1155/2016/9895176

Figure Lengend Snippet: BDL in HC fed mice increases apoptosis. Tissue was obtained from Sham and three days after BDL animals, under (a-b) standard Chow control diet (Chow) or (c-d) high cholesterol (HC) diet, and apoptosis was assayed by (e) TUNEL immunohistochemistry; (e) quantification of positive TUNEL cells or by (f) caspase 3 activity. Images are representative from at least four independent mice. Original magnification: 100x. ∗ p < 0.01 versus Chow fed mice, # p < 0.01 versus 3 days Chow fed mice, and & p < 0.01 versus HC fed mice.

Article Snippet: For each reaction, caspase 3 synthetic fluorogenic tetrapeptide substrate (Ac-DEVD-AMC) was added (BD Pharmingen) and then the tissue lysate; after that samples were incubated for one hour.

Techniques: TUNEL Assay, Immunohistochemistry, Activity Assay