TiterTACS™ Colorimetric Apoptosis Detection Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add Proteinase K solution and incubate for 15 minutes at room temperature. Add Labeling Reaction mix and incubate at 37°C for 1 hour. Add TACS-Sapphire at room temperature..

Protocol tips
Add Proteinase K solution and incubate for 15 minutes at room temperature. Add Labeling Reaction mix and incubate at 37°C for 1 hour. Add TACS-Sapphire at room temperature..

Publication protocol

For ADAM12 augmentation and knockdown in vitro, plasmid containing full-length ADAM12 cDNA or shRNA targeting ADAM12 was transfected into HUVECs using a high-efficiency primary cell-transfection reagent (Cytofect EC transfection kit; Cell Applications), following the manufacturer's protocol. Briefly, cells were cultured to ∼75% confluence in a six-well plate. DNA plasmid (3 μg) was mixed with 2.5 μl Cytofect-2 and 2.5 μl polyethylene reagents and incubated at 37°C for 30 min. This plasmid DNA and transfection reagent complex mixture was added to ECs for 1 h and then replaced with ECGM with 10% serum. Cells were used in subsequent experiments, 24 h after transfection. EC proliferation, apoptosis assay, and in vitro angiogenesis were assessed, as described previously (16). Briefly, proliferation was assessed under baseline conditions (ECGM with 10% serum) using tetrazolium dye incorporation (BioVision, Milpitas, CA), 48 h post-transfection, following the manufacturer's protocol. Apoptosis was assessed in transfected cells, 24 h post-transfection, followed by 24 h exposure to simulated ischemia using the terminal deoxynucleotidyltransferase (TdT)-mediated 2′-deoxyuridine 5′-triphosphate nick end-labeling assay (TiterTACS; Trevigen, Gaithersburg, MD), following the manufacturer's protocol. Nuclease-treated wells were used as positive controls, whereas wells without terminal TdT were used as negative controls. For the angiogenesis assay, the cells were plated on growth factor-enriched matrigel (Catalog #356234; BD Biosciences), 48 h post-transfection, and grown in simulated ischemia for 6 h. Each condition was performed in quadruplicate, and representative pictures were taken from each well at 100× magnification. A blinded observer counted the number of complete tubes, which was expressed as tubes/mm2. All experiments described above were repeated at least three times.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing TUNEL assay cell type - Mouse endothelial cells using TiterTACS™ Colorimetric Apoptosis Detection Kit from Bio-Techne.

Manufacturer protocol

Download the product protocol from Bio-Techne for TiterTACS™ Colorimetric Apoptosis Detection Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing TUNEL assay cell type - Mouse endothelial cells using TiterTACS™ Colorimetric Apoptosis Detection Kit from Bio-Techne. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.