In situ cell death kit

Manufactured by Roche

Sourced in United States, Germany, Switzerland

The In situ cell death kit is a laboratory product that enables the detection and visualization of cell death in tissue samples. The kit provides the necessary reagents and protocols to perform this analysis.

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Lab products found in correlation

Lsm 780 confocal microscope, by Zeiss (3 mentions) N octyl glucopyranoside, by Merck Group (2 mentions) Fv 1000 laser scanning confocal biological microscope, by Olympus (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Crude trypsin, by Thermo Fisher Scientific (2 mentions) Dapi karyotyping kit, by Genmed Scientifics (1 mentions) Facscalibur, by BD (1 mentions) β catenin, by Cell Signaling Technology (1 mentions) Fortessa flow cytometer, by BD (1 mentions) Annexin 5 and pi, by BD (1 mentions) Annexin 5, by BD (1 mentions) Axio imager 2 microscope, by Zeiss (1 mentions) Lysotracker green, by Thermo Fisher Scientific (1 mentions) Cellrox deep red, by Thermo Fisher Scientific (1 mentions) Lysotracker red, by Thermo Fisher Scientific (1 mentions) Fv1000, by Olympus (1 mentions) Microtome, by Thermo Fisher Scientific (1 mentions) Eclipse 50i microscope, by Nikon (1 mentions) Digital camera, by Canon (1 mentions) Lab tek chamber slide, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

One-step in situ cell death detection kit TUNEL staining kit (In Situ Cell Death Detection Kit) Fluorescent In Situ Cell Death Detection Kit In situ cell death determination kit Fluorescein In Situ Cell Death Detection Kit In Situ Cell Death Detection Kit (TUNEL fluorescence FITC kit; In situ cell death detection kit I In Situ Cell Death Detection Kit with fluorescein-dUTP In Situ Cell Death Fluorescein Kit TUNEL in situ cell death detection kit AP

48 protocols using in situ cell death kit

Evaluation of Apoptosis Induction and Caspase-3 Expression

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Early stages of apoptosis induction were evaluated using Annexin V binding, an indicator of early apoptosis (Huppertz, Frank et al., 1999 (link)). Annexin V-PE or-FITC (Pharmingen, San Diego, CA) staining was combined with staining for cell phenotype. Measurements of late-stage apoptosis were determined by quantitation of DNA strand breaks (Nichols, Niles et al., 2001 (link)) using the TUNEL Assay (In Situ Cell Death Kit, Boehringer Mannheim, Mannheim, Germany). In a subset of experiments, the apoptosis was confirmed after cell sorting by demonstration of DNA fragmentation (Gavrieli, Sherman et al., 1992 (link)).
Caspase-3 level was evaluated by staining 10⁶ sham-exposed or virus-exposed PBMC with 20 ul of PerCP conjugated anti-CD3 antibody (44). After 30 minutes the cells were washed and fixed in 2% PAF for 2 hours. The CD3+ stained cells were permeabilized for 10 minutes using 0.6% n-octyl glucopyranoside (Sigma Chemical Co.) and then stained for caspase-3 expression with 20 ul of FITC-conjugated rabbit anti-active caspase-3 monoclonal antibody (clone C-92-605) (BD Pharmingen). After 30 minutes cells were washed and analyzed immediately.
Anti-NA antibodies (N1 or N3 - NIAID Reference Reagents), or the NA inhibitors 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid (5mM) or N-methyldeoxynojirimycin (5mM) were added to cells in a subset of experiments to inhibit NA activity.

Nichols J.E., Niles J.A., Fleming E.H, & Roberts NJ J.r. (2019). The Role of Cell Surface Expression of Influenza Virus Neuraminidase in Induction of Human Lymphocyte Apoptosis. Virology, 534, 80-86.

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Quantifying Apoptosis via TUNEL Assay

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Apoptosis was determined by quantitation of DNA strand breaks using the TUNEL Assay (In Situ Cell Death Kit, Boehringer Mannheim) [Nichols et al. 2001 (link)]. Cell phenotype was established by cell surface labeling with monoclonal antibodies prior to the TUNEL reaction. After staining, cells were fixed in 2% PAF and permeabilized for 10 minutes using 0.6% n-octyl glucopyranoside (Sigma Chemical Co.). The TUNEL reaction was carried out as described in the manufacturer’s instructions.

Fleming E.H., Ochoa E.E., Nichols J.E., O'Banion M.K., Salkind A.R, & Roberts NJ J.r. (2017). Reduced activation and proliferation of human lymphocytes exposed to respiratory syncytial virus compared to cells exposed to influenza virus. Journal of medical virology, 90(1), 26-33.

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Tumor Apoptosis by TUNEL Assay

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The cell apoptosis of the tumor samples was measured by TUNEL assay using an in situ cell death kit (Roche) by following the manufacture's protocol. The cell nucleuses were counterstained by DAPI (Sigma) reagent. Positive cells were visualized by FV-1000 laser scanning confocal biological microscopes (Olympus).

Zhou X., Ren Y., Kong L., Cai G., Sun S., Song W., Wang Y., Jin R., Qi L., Mei M., Wang X., Kang C., Li M, & Zhang L. (2015). Targeting EZH2 regulates tumor growth and apoptosis through modulating mitochondria dependent cell-death pathway in HNSCC. Oncotarget, 6(32), 33720-33732.

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Annexin V Apoptosis Assay and TUNEL Analysis

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Parental and transfected cells in the log phase of growth were harvested and collected. For the Annexin V assay, the annexin V-Cy3-labeled Apoptosis Detection Kit (Abcam, USA) was used. The apoptotic cells were detected and quantified using FACSCalibur (Becton Dickinson, USA). The data obtained were analyzed using CellQuest software. The apoptotic cell death in the tumor specimens of nude mouse models from in vivo study was examined by TUNEL method using in situ cell death kit (Roche, USA). Nuclei were counterstained with DAPI karyotyping kit (Genmed, China) and visualized using FV-1000 laser scanning confocal biological microscope and analyzed by IPP5.1 (Olympus, Japan).

Li W., Dong S., Wei W., Wang G., Zhang A., Pu P, & Jia Z. (2016). The role of transcriptional coactivator TAZ in gliomas. Oncotarget, 7(50), 82686-82699.

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Histological and Immunohistochemical Analysis of Colonic Inflammation and Dysplasia

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Histological analysis was performed as previously described [20 (link)]. In brief, fresh colon pieces were fixed in 4% Paraformaldehyde solution and embedded in paraffin. Five-micron sections of paraffin-embedded colons were stained with hematoxylin and eosin (H&E) and analyzed for severity of inflammation and dysplasia. For immunohistochemical analysis, standard immunohistochemical procedures were performed using the following antibodies: β-catenin (Cell Signaling Technology; 8480) and Ki67 (Abcam, ab16667). Intratumoral apoptotic cells were detected in paraffin-embedded colon samples with the In Situ Cell Death Kit (Roche, 11684817910) according to the manufacturer’s instructions. After the immunohistochemical staining, slides were read under microscope and fields were randomly selected for further analysis. Images were taken and the number of positive cells in each field per high-power field within each tumor from WT and Tipe^−/− tumor-bearing mice were counted. At least three fields for each mouse were used for quantification, with the animal number of five for each group.

Lou Y., Tian X., Sun C., Song M., Han M., Zhao Y., Song Y., Song X., Zhang W., Chen Y.H, & Wang H. (2022). TNFAIP8 protein functions as a tumor suppressor in inflammation-associated colorectal tumorigenesis. Cell Death & Disease, 13(4), 311.

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TUNEL Assay for Apoptosis Detection

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The in situ cell death kit (Roche Molecular Biochemicals, Mannheim, Germany) based on TUNEL technology was performed to detect individual apoptotic cells in formalin fixed tissue sections. Dewax and rehybrate tissue section according to standard protocols. Endogenous peroxidase activity was blocked by incubated in 0.3% hydrogen peroxide for 10 min. Slides were made permeable with pereabilisation on solution. After washing, slides were incubated with TUNEL reaction mixture for 60 min at 37℃ in a humified atmosphere in the dark. The negative control was incubated with label solution (without forminal transferase) instead of TUNEL reaction mixture. After washing, samples can be analyzed in a drop of PBS under a fluorescence microscope. Use an excitation wave length in the range of 450-500nm and detection in the range of 515-565nm.

Zhao H.J., Ren L.L., Wang Z.H., Sun T.T., Yu Y.N., Wang Y.C., Yan T.T., Zou W., He J., Zhang Y., Hong J, & Fang J.Y. (2014). MiR-194 Deregulation Contributes To Colorectal Carcinogenesis via Targeting AKT2 Pathway. Theranostics, 4(12), 1193-1208.

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Apoptosis Quantification via TUNEL and Annexin V

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For the TUNEL assay, an In Situ Cell Death Kit (Roche) was used according to the manufacturer's recommendations. For Annexin V and PI staining, cells were stained with 50 µg/ml PI and Annexin V (BD Bioscience) in Annexin V buffer. The cells were analyzed by Fortessa flow cytometer (BD Biosciences).

Liu Z.Y., Zheng M., Li Y.M., Fan X.Y., Wang J.C., Li Z.C., Yang H.J., Yu J.M., Cui J., Jiang J.L., Tang J, & Chen Z.N. (2019). RIP3 promotes colitis-associated colorectal cancer by controlling tumor cell proliferation and CXCL1-induced immune suppression. Theranostics, 9(12), 3659-3673.

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Apoptosis Assessment in Testis Tissue

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At Pnd31, testes from control and MET groups (n = 4/group) were fixed in formalin 10% and embedded in paraffin. Two nonconsecutive testis sections (3–5 μm) were blocked with a horse serum dilution. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed as previously described [33 (link)]. The In Situ Cell Death Kit (Roche Applied Science, Indianapolis, IN, USA) was used. For negative controls, the TUNEL reaction mixture without terminal transferase was used. Cell nuclei were stained with DAPI. Epifluorescence microscopy was performed using an Axio Imager 2 microscope (Zeiss, Germany) coupled with an Axiocam 202 monochrome camera (Zeiss). Histological sections were counted and averaged per animal. A minimum of 400 transversal seminiferous tubules were selected in each tissue section. The number of TUNEL-positive cells per round seminiferous tubule was quantified in each tissue section using Image J version 1.53k software (NIH, Bethesda, MD, USA). The incidence of apoptosis was also reported as the apoptotic index (AI). The AI was calculated as the percentage of seminiferous tubules showing three or more TUNEL-positive cells.

Rindone G.M., Dasso M.E., Centola C.L., Sobarzo C.M., Galardo M.N., Meroni S.B, & Riera M.F. (2024). Effect of Metformin on Sertoli Cell Fatty Acid Metabolism and Blood–Testis Barrier Formation. Biology, 13(5), 330.

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Retinal Microvascular Degeneration Analysis

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The retina from formalin-fixed eyes was rinsed overnight with running water, and was incubated at 37°C with 3% crude trypsin (Invitrogen-Gibco, Grand Island, NY, USA) containing 200 mM sodium fluoride for 45 to 70 minutes. After gently brushing away the neuro-retinal tissue, trypsin-digested microvasculature mounted on a glass slide was stained with terminal deoxyribonucleotide transferase-mediated dUTP nick-end labelling (TUNEL) stain (In Situ Cell Death kit; Roche Molecular Biochemicals, Indianapolis, IN, USA). The microvessels were then rinsed, and stained with periodic acid Schiff-hematoxylin to count the degenerative capillaries [21 (link), 24 (link)].

Kowluru R.A., Mishra M., Kowluru A, & Kumar B. (2016). Hyperlipidemia and the development of diabetic retinopathy: Comparison between type 1 and type 2 animal models. Metabolism: clinical and experimental, 65(10), 1570-1581.

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Cell Death Detection in Tetrapod Limb Development

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Cell death was detected using LysoTracker, Nile Blue, or TUNEL, as previously described [17 (link), 26 (link), 27 (link)]. LysoTracker labels the increased lysosomal activity detected in dying cells and around phagocytosed cell debris [28 (link), 29 (link)] and has been used to identify the cell death in limb buds of various tetrapods, including X. laevis and coqui frogs [17 (link), 30 ]. Briefly, tadpoles at stages 32–37 were incubated with 0.5 μM LysoTracker Red (Thermo Fisher Scientific, Waltham, MA) in PBS+ (PBS pH 7.4, 9 mM CaCl₂, 3.3 mM MgCl₂) for 2 hours, washed and photographed with na LSM780 confocal microscope (Zeiss). Some of the LysoTracker Red-stained tadpoles were then stained with 0.01% Nile blue in filtered water for 20 minutes, washed, and photographed. For TUNEL staining, hindlimb buds from stage 36 tadpoles were cryosectioned at 8–12 μm as described [17 (link)], and stained using TUNEL Mix (In situ Cell Death Kit, Roche) according to the manufacturer’s protocol. For detection of cell death and reactive oxygen species, tadpoles at stage 36–37 were incubated with 0.5 μM LysoTracker Green and with 2 μM CellROX Deep Red (Invitrogen) in PBS+ for 2 hours, washed, and photographed with an LSM780 confocal microscope (Zeiss).

Ono S.F., Cordeiro I.R., Kishida O., Ochi H, & Tanaka M. (2023). Air–breathing behavior underlies the cell death in limbs of Rana pirica tadpoles. Zoological Letters, 9, 2.

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