In situ cell death detection kit

After GSTCD siRNA knockdown in HBECs the cell proliferation rate, total cell number and apoptosis were quantified at different time intervals using the CyQUANT® NF proliferation assay, Click-iT® EdU microplate assay and Apoptosis was measured using the In situ cell death detection kit (Roche).
The CyQUANT® NF proliferation assay measures total cell number utilising a cell permeable fluorescent DNA binding dye applied to the cells at the experimental end-point. Click-iT® EdU microplate assay labels actively proliferating cells by incorporating the thymidine analogue 5-ethynyl-2-deoxyuridine (EdU) into DNA during cell replication, followed by antibody based signal amplification resulting in a fluorescence reading. Apoptosis was measured using the In situ cell death detection kit (Roche). In this assay the green fluorescent is an indicator of broken down DNA strands. The HBECs were seeded on 8-well chamber slides and transfected with the siRNA as previously described, cells were then fixed and the ratio of green apoptotic cells to the total number of blue DAPI stained cells were compared.

The apoptosis-related proteins were evaluated by TUNEL using the In Situ Cell Death Detection Kit (Boehringer Mannheim, Indianapolis, IN) according to instructions. The percentage of apoptotic cells was calculated by counting 200 cells in 5 fields in each experiment. Five high magnification fields were randomly selected from the border zone of infarcted myocardium in each section and the number of positive cells was recorded. Apoptosis Index (AI%) = Apoptotic cell nuclei / total cell nuclei × 100%^{44 (link)}.

To determine myocardial apoptosis, cardiac tissues from the MI group, sham group, MI + meto group, MI + terbu group, and MI + meto + terbu group were perfused first with 0.9% NaCl for 5 min and then with 4% paraformaldehyde in PBS (pH = 7.4) for 20 min. Ischemic regions were cut to 5 mm thickness at a level of left ventricular long axis and further fixed in 4% paraformaldehyde in PBS for 24 h at room temperature. Fixed tissues were embedded in a paraffin block and two slides at 5 μm thickness were cut from each tissue block [33 (link)]. The slides were processed for a TUNEL assay. An In Situ Cell Death Detection Kit (Boehringer Mannheim, USA) was used according to the manufacturer's instructions. Briefly, the slides were treated with H₂O₂ and incubated with the reaction mixture containing TdT (terminal deoxyribonucleotide transferase) and FITC-conjugated dUTP at 4°C overnight. One part of labeled DNA was visualized with fluorescent microscopy. Another part was incubated subsequently with alkaline phosphatase (AP) conjugated anti-FITC antibody for 1 h at 37°C and then visualized using AP-Red as the chromogen. Finally, the labeled DNA was detected with medical image analysis system. For negative control, TdT was omitted from the reaction mixture. Assays were performed in a blinded manner.

Embryos at the one- or two-cell stage were injected with YY1-MO (25 ng) or control-MO (25 ng) or YY1-MO (25 ng) plus PSR mRNA (1 ng), harvested at 18.5 hpf, fixed with 4 % paraformaldehyde in PBS (pH 7.4) at room temperature for 30 min, stained with acridine orange (1 μg.ml⁻¹; 3–5 min), washed twice in PBS, and evaluated under a fluorescence microscope (using a 488-nm filter for excitation and a 515-nm long-pass filter for detection) [44 (link)]. For the TdT-dUTP labeling step, the embryos were fixed in paraformaldehyde at the end of the incubation period (12 and 24 hpf), dechorionated, incubated in blocking solution (0.1 % H₂O₂ in methanol, 30 min, room temperature), rinsed with PBS, incubated on ice in 0.1 % Triton X-100 in 0.1 % sodium citrate for 30 min to increase permeability, rinsed twice with PBS, treated with TUNEL reaction mixture (50 μl; in-situ cell death-detection Kit, Boehringer Mannheim), incubated in a humidified chamber for 60 min at 37 °C, and evaluated for fluorescence-positive apoptotic cells under a fluorescence microscope equipped with a spot II CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI).