Cells were seeded and treated with the indicated drugs for 48 h, detached from the plates with Trypsin Solution without EDTA (Beyotime Biotechnology, Shanghai, China) and washed with cold PBS. The induction of apoptosis was measured by FCM using the Annexin V-PE/7-AAD Apoptosis Detection Kit (Yeasen Biotech, Shanghai, China). And for detection of DNA breakage, a TUNEL assay was performed following the protocol provided by the manufacturer (Yeasen Biotech).
Tunel assay
Manufactured by Yeasen
Sourced in China
The TUNEL assay is a laboratory technique used to detect and quantify apoptosis, a type of programmed cell death. The assay works by labeling the fragmented DNA that is characteristic of apoptotic cells, allowing for the identification and measurement of these cells within a sample.
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5 protocols using tunel assay
1
Apoptosis Quantification by FCM and TUNEL
2
TUNEL Assay for Apoptotic Cells
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Apoptotic cells were examined with TUNEL assay (Yeasen) according to the manufacturer’s instructions. In brief, brain slices were digested for 10 min by proteinase K (20 μg/ml) at room temperature after IF staining. After washing twice with PBS, brain slices were incubated with equilibration buffer for 30 min at room temperature, and subsequently with Alexa Fluor 488-12-dUTP Labeling Mix for 60 min at 37 °C. After washing with PBS for three times, brain slices were stained with DAPI (2 μg/ml, Roche, Basel, Switzerland) before being mounted under coverslips.
3
Apoptosis Quantification in RASMCs
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Measurement of apoptotic RASMCs was determined by flow cytometry-based Annexin V-PE staining (C1065S, Beyotime, China) and Hoechst (C1028, Beeyotime, China). The double-negative cells (viable), Annexin V single-positive cells (apoptosis), and double-positive cells (necrosis) were analyzed using FlowJo Software (V10.0.7, USA). Apoptotic cells in carotid arteries were assessed using the TUNEL assay (40307ES20, YEASEN, China). The number of TUNEL-positive cells was counted in 10 randomly selected fields in each sample under ×400 magnification by using Image J.
4
Evaluating LPS-Induced Apoptosis in PMVECs
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The TUNEL assay (40307ES50, Yeasen Biotechnology) was employed to evaluate LPS-induced apoptosis in PMVECs. Initially, following enzymatic digestion, PMVECs were collected for slide preparation. Post-LPS treatment, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min and permeabilized with 0.3% Triton X-100 for 5 min at room temperature. Subsequently, PMVECs were incubated with 100 µL of Equilibration Buffer for 30 min at room temperature, followed by incubation with TdT incubation buffer for 1 h at 37 °C in a humidified chamber away from light. After washing with PBS, the nuclei were stained with 4’,6-Diamidino-2-Phenylindole (DAPI) for 5 min. The enumeration of TUNEL-positive cells was conducted using a fluorescence microscope, and the proportion of apoptotic cells was quantitatively analyzed.
5
Hippocampal Apoptosis in Cardiac Arrest
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Rats were sacrificed 24 h after the ROSC. Hippocampal tissues were carefully removed and immersed in paraformaldehyde overnight. Then, all tissues were embedded in paraffin and sliced into 5-μm sections. For histologic assessment, sections were stained with 0.01% Toluidine blue (Sinopharm Group, Shanghai, China) and mounted. Terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay (Yeasen Biotech, Shanghai, China) was performed using an in-situ apoptosis detection kit, as per the manufacturer's instruction. Three fields of vision in the hippocampal CA area were randomly selected, and the viable neurons and TUNEL-stained neuronal cells were counted under an optical microscope.
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