Deadend fluorometric tunel kit

DNA fragmentation was evaluated by Dead-End^TM Fluorometric TUNEL kit (Promega) [6 (link)], and by Flow cytometry quantification of hypodiploid cells (sub-G1 fraction), as described [6 (link)].

For liver apoptosis assay, terminal deoxynucleotidyl transferased UTP nick end labeling (TUNEL) staining was performed using a DeadEnd^TM Fluorometric TUNEL kit (Promega, Madsion, WI). Tissue sections were stained for nuclei (4′,6-diamidino-2-phenylindole (DAPI) staining) and apoptotic nuclei (TUNEL staining) and analyzed using a confocal laser scanning microscope. For heart tissues, TUNEL staining was performed using in situ cell death detection Kit (Roche Applied Science, Indianapolis, IN) as described previously. To distinguish cardiomyocyte from non-cardiomyocyte nuclei, we used triple stain for nuclei (DAPI staining), apoptotic nuclei (TUNEL staining), and cardiomyocytes (α-Actinin staining), and analyzed the stained sections using confocal microscopy. A minimum of ~10 high power fields with ~2000 nuclei/field were counted for each sample.

DNA fragmentation was evaluated by the DeadEnd^TM Fluorometric TUNEL kit (Promega) according to the manufacturer's directions and flow cytometric quantification of hypodiploid cells (sub-G₁ fraction). For quantification of hypodiploid events, 10⁵ cells/well were transfected for 48 h as described above. Staurosporine (STP, Sigma) was used at a concentration of 5 μm as a positive control of apoptosis. Cells were harvested and centrifuged at 600 × g for 5 min. Pellets were suspended in 100% ethanol and stored at −20 °C for 24 h. Cells were then treated with 1 mg/ml RNase A for 1 h at RT. PI was added, and samples were subjected to flow cytometry.

DNA fragmentation was determined by the Dead End TM Fluorometric TUNEL kit (Promega), according to manufacturer’s directions. At least ten fields per sample were analyzed under an Olympus BX-53 fluorescence microscope. Phosphatidylserine exposure was determined by Annexin-V binding with the APOtarget kit (Thermo Fisher Scientific), according to manufacturer’s directions, co-stained with PI and analyzed by Flow cytometry on a BD Biosciences (San Jose, CA, USA) FACS Canto Flow Cytometer.

Death of cardiomyocytes at the border region of the infarcted heart was detected using the DeadEnd^TM Fluorometric TUNEL kit (Promega, Madison, WI, USA).
H9C2 cells were treated with conditioned medium from Card9-overexpressing and control RAW264.7 cells for 48 h, and then their apoptosis was evaluated using TUNEL staining.

Ovaries of WPP stage were selected from strictly white pupae. Adult ovaries were taken from day 3 females. All ovaries were dissected in phosphate buffered saline (PBS), fixed for 10 min in 4% paraformaldehyde at room temperature, washed three times in PBS containing 0.1% Triton X-100 (PBT) (each for 15 min), and blocked in 5% normal goat serum for 1 h. Ovaries were incubated with primary antibodies diluted in PBT overnight at 4 °C. Then, ovaries were washed three times in PBT and incubated with secondary antibodies at room temperature for 1 h. After washing three times again with 0.1% PBT, ovaries were finally mounted in PBS/glycerol medium with DAPI as described previously [47 (link)]. The primary antibodies used were rabbit anti-Lola (a gift from Professor Lei Zhang) (1:500) and mouse anti-En (4D9, DSHB, IA, USA) (1:50). The secondary antibodies were DyLight 594 conjugated goat anti rabbit (#A23440, Abbkine, Beijing, China) (1:1000) and DyLight 488 conjugated goat anti mouse (#A23210, Abbkine, Beijing, China) (1:1000). TUNEL assay was performed using the DeadEnd Fluorometric TUNEL kit (Promega, WI, USA) following the manufacturer’s instructions. All images were collected on a Zeiss LSM 710 Confocal Microscope (Thornwood, NY, USA).

Sections were sectioned at 10 um, as in staining protocols above. TUNEL staining used the Promega DeadEnd Fluorometric TUNEL kit, with which cells were briefly permeabilized using 5% Triton X-100 in PBS; washed in PBS; equilibrated in 100 μl eq. buffer at room temperature for 10 minutes; labelled with TdT mix for 50 minutes at 37°C shielded from light—the reaction stopped with 2-time SSC (15 minutes)—washed 3 times in PBS/0.3% Triton/2% BSA; and finally washed in PBS (5 minutes each). Slides were then blocked, and immunostained, as above.