Cell apoptosis was measured using a caspase-3 activity assay kit (Beyotime Institute of Biotechnology) and an in situ cell death detection kit (Beijing Solarbio Science & Technology Co., Ltd.), according to the supplier's instructions. For the caspase-3 assay, cells (1×104) were seeded into 96-well white opaque plates and allowed to adhere overnight. Following different treatments, the cells were lysed and incubated with 2 mM Ac-DEVD (Asp-Glu-Val-Asp)-pNA in reaction buffer at 37°C for 1 h. The absorbance values at 405 nm were detected using an EL ×800 strip reader (BioTek Instruments Inc.). For the TUNEL assay, after fixation with 4% paraformaldehyde for 30 min, the cells were incubated with TUNEL buffer for 1 h at 37°C and then rinsed three times with PBS. The number of TUNEL-positive cells and the total number of cells in five different random fields were counted under a magnification of ×400 using a light microscope (Olympus Corporation). The rate of apoptotic cells was expressed as a percentage over the total cells.
In situ cell death detection kit
Manufactured by Solarbio
Sourced in China
The In Situ Cell Death Detection Kit is a laboratory product designed to detect and quantify cell death in biological samples. It enables the identification of apoptotic, necrotic, and autolytic cells using a range of techniques.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 3 activity assay kit, by Beyotime (1 mentions)
Eclipse te2000 e, by Nikon (1 mentions)
Hematoxylin, by Wuhan Servicebio Technology (1 mentions)
Nisin staining solution, by Wuhan Servicebio Technology (1 mentions)
Tnf α, by Bioswamp (1 mentions)
Dab concentrated kit, by Solarbio (1 mentions)
Pentobarbital sodium, by Merck Group (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Neutral resin, by Sinopharm (1 mentions)
Primescript 2 rtase, by Takara Bio (1 mentions)
Bca protein concentration assay kit, by Solarbio (1 mentions)
Ifn γ, by Bioswamp (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
7 protocols using in situ cell death detection kit
1
Quantifying Cellular Apoptosis via Caspase-3 and TUNEL
2
Apoptosis Evaluation via TUNEL Assay
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Terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL) was conducted to evaluate apoptosis by using the In Situ Cell Death Detection Kit (Solarbio). Paraffin sections were used for TUNEL staining.16 After deparaffinization, the paraffin sections were incubated with 10 μg/ml of proteinase K and then rehydrated for 15 min. Fresh TUNEL reaction mixtures were added to sections, which were incubated for 60 min in darkness and at 37°C. After they were washed, the cell nuclei were stained with 0.1 μg/ml 4′,6‐diamidino‐2‐phenylindole (DAPI). The sections were analysed with a drop of PBS by fluorescence microscopy (Canon).
3
Brain Apoptosis Assessment by TUNEL Staining
4
Apoptosis Analysis in Myocardial Tissues
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Cell apoptosis in the myocardial tissues of model rats was analysed by TUNEL assays using an In Situ Cell Death Detection Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Specifically, the heart tissues were fixed in 4% paraformaldehyde for 48 h and then embedded in paraffin. Then, 4 μm-thick serial sections were made, deparaffinized, dehydrated in graded alcohol and stained with the reagents in the In Situ Cell Death Detection Kit. Apoptotic nuclei stained by TUNEL were brownish yellow. Finally, the apoptotic cells were observed under a confocal laser scanning microscope.
5
Investigating 7,8-DHF Neuroprotective Effects
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Para-chlorophenylalanine (PCPA) was purchased from Shanghai Genye. The 0.9% NaCl solution was adjusted to a weakly basic pH (pH 7.0–8.0) with NaOH and HCl to prepare a PCPA suspension (5% Tween-80) for use in this study. 7,8-Dihydroxyflavone (7,8-DHF) was purchased from MCE, pentobarbital sodium was purchased from Sigma Merck, dimethyl sulfoxide (DMSO) was purchased from Solepipe, and PEG300 was purchased from MCE. Eosin was purchased from Solarbio, hematoxylin was purchased from Servicebio, nisin staining solution was purchased from Servicebio, neutral resin was purchased from Shanghai Sinopharm, the In Situ Cell Death Detection Kit, the DAB concentrated kit was purchased from Solarbio, hematoxylin was purchased from Servicebio, the Marker was purchased from Helix, and the BCA protein concentration assay kit was purchased from Solarbio. ELISA kits for interleukin-1β (IL-1β), prostaglandins D2 (PGD2), melatonin (MT), corticosterone (CORT), IL-6, IL-10, tumor necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), and brain-derived neurotrophic factor (BDNF) were purchased from Bioswamp. TRIzol was purchased from Ambion, and PrimeScript II Rtase was purchased from TAKARA. Antibodies against Bcl-2, Bax, Bad, Caspase-3, TrKB, PI3K, Akt, cAMP, CREB, BDNF, GAPDH, p-TrKB, p-Akt and p-CREB, as well as goat anti-rabbit IgG, were purchased from CST.
6
Chondrocyte Apoptosis Detection by TUNEL
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Apoptotic chondrocytes were measured by TUNEL staining using an In Situ Cell Death Detection kit, according to the manufacturer's instructions. The chondrocytes (1×105) were seeded in a 6-well plate and treated with SFN (50 µM) or H2O2 (50 µM) for 24 h at 37°C, fixed in 4% paraformaldehyde for 45 min at 37°C, incubated with 0.5% Triton X-100 for 15 min at 37°C and washed using PBS for 5 min. Finally, cells were stained using the In Situ Cell Death Detection kit for 60 min at 65°C, after which the nuclei were stained with DAPI for 1 min at 37°C (Beijing Solarbio Science & Technology Co., Ltd.). In total, 25 fields of each slide were randomly selected and images were observed using a fluorescence microscope (magnification, ×100; scale bar, 50 µm; Olympus Corporation).
7
Apoptosis Analysis of Tumor Tissue
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Tumour tissue was fixed in 4% formaldehyde for 24 h, embedded in paraffin and then sectioned (5.0 mm). Sections were stained with haematoxylin and eosin for histological analysis. The TUNEL assay was performed with an In Situ Cell Death Detection kit (Solarbio, Beijing, China) according to the manufacturer's protocols.
Five fields of view (200×) were randomly selected on each slide, and the number of total cells and TUNEL-positive cells were counted. The apoptosis rate was calculated based on the formula: the apoptosis rate (%) = the number of TUNEL-positive cells/the number of total cells ×100%.
Five fields of view (200×) were randomly selected on each slide, and the number of total cells and TUNEL-positive cells were counted. The apoptosis rate was calculated based on the formula: the apoptosis rate (%) = the number of TUNEL-positive cells/the number of total cells ×100%.
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