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Deadend fluometric tunel system

Manufactured by Promega
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The DeadEnd Fluometric TUNEL System is a lab equipment product that detects and quantifies apoptosis, a form of programmed cell death, in samples. It utilizes the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay to label DNA strand breaks, allowing for the identification and analysis of apoptotic cells.

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Lab products found in correlation

Zen 2, by Zeiss (2 mentions) Lsm 880 laser confocal microscope, by Zeiss (2 mentions) Fluorescence microscopy, by Nikon (1 mentions) Mtt assay, by Roche (1 mentions)

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DeadEnd TUNEL System TUNEL) system

5 protocols using deadend fluometric tunel system

1

Apoptosis Quantification in Cardiac Tissue

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Terminal dUTP nick end-labeling (TUNEL) nuclei were visualized at X400, as previously described [32 (link),33 (link)]. In brief, apoptotic cells in left ventricular (LV) sections were detected by labelling of nuclear DNA strand breaks with the DeadEnd Fluometric TUNEL System (Promega, Madison, WI, USA). The apoptotic index was calculated as TUNEL + nuclei × 100/DAPI + nuclei.
Tsoporis J.N., Amatullah H., Gupta S., Izhar S., Ektesabi A.M., Vaswani C.M., Desjardins J.F., Kabir G., Teixera Monteiro A.P., Varkouhi A.K., Kavantzas N., Salpeas V., Rizos I., Marshall J.C., Parker T.G., Leong-Poi H, & dos Santos C.C. (2023). DJ-1 Deficiency Protects against Sepsis-Induced Myocardial Depression. Antioxidants, 12(3), 561.
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2

Apoptosis Detection via TUNEL Assay

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Apoptosis detection was performed according to the instruction of the DeadEnd™ Fluometric TUNEL system (Promega, WI, USA, Cat# G3250). Tissue sections were post-fixed in 4% PFA, followed by 20μg/ml Proteinase K treatment to prevent desorption. Sections were then rinsed in Equilibration buffer for 10 min at room temperature and subsequently incubated in Terminal deoxynucleotidyl Transferase (TdT) reaction mix according to manufacturer’s instructions. Finally, the reaction was terminated by high concentration of salt solution, like 2X Saline Sodium Citrate (SSC). After rinsed three times in PBS, sections were mounted in Mowiol Mounting Medium.
Xu X., Yu Q., Fang M., Yi M., Yang A., Xie B., Yang J., Zhang Z., Dai Z, & Qiu M. (2019). Stage-specific regulation of oligodendrocyte development by Hedgehog signaling in the spinal cord. Glia, 68(2), 422-434.
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3

Quantification of Osteoblast Apoptosis

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In vivo osteoblast apoptosis was quantified by using the DeadEnd™ Fluometric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, vertebral sections embedded in methyl methacrylate were deplastified using xylene and monoethanolamine. The slides were washed with acetone, rehydrated with decreasing concentrations of ethanol, and rinsed in distilled water. The slides were then fixed in 4% formaldehyde, permeabilized with proteinase K solution (20 μg/mL), fixed again and equilibrated with equilibration buffer (100 μL). The samples were then labeled with terminal deoxynucleotidyl transferase (TdT) and incubated for 60 min in a humidified chamber and then counterstained with DAPI. The apoptotic osteoblasts were identified by green flourescence using confocal fluorescence microscopy (Zeiss LSM 880 laser confocal microscope; Zeiss, Inc.) using the following settings: objective = EC plan‐Neofluar 40×/1.3 oil; wavelength of excitation 405 nm for DAPI and 488 nm for TUNEL staining. We obtained 25 tiled images (sample in five‐frame stack with 3.5‐μm interval). The stacks were converted to orthogonal projections and the osteoblasts were counted using Zen 2.3 software (Zeiss, Inc.).
Palmieri M., Kim H., Gomez‐Acevedo H., Que X., Tsimikas S., Jilka R.L., Manolagas S.C., Witztum J.L, & Ambrogini E. (2020). A Neutralizing Antibody Targeting Oxidized Phospholipids Promotes Bone Anabolism in Chow‐Fed Young Adult Mice. Journal of Bone and Mineral Research, 36(1), 170-185.
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4

Quantification of Osteoblast Apoptosis by TUNEL

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In vivo osteoblast apoptosis was quantified by using the Dead-End™ Fluometric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, vertebral sections embedded in methyl methacrylate were deplastified using xylene and monoethanolamine. The slides were washed with acetone, rehydrated with decreasing concentrations of ethanol, and rinsed in distilled water. The slides were then fixed in 4% formaldehyde, permeabilized with proteinase K solution (20 μg/mL), fixed again and equilibrated with equilibration buffer (100 μL). The samples were then labeled with terminal deoxynucleotidyl transferase (TdT) and incubated for 60 min in a humidified chamber and then counterstained with DAPI. The apoptotic osteoblasts were identified by green flourescence using confocal fluorescence microscopy (Zeiss LSM 880 laser confocal microscope; Zeiss, Inc.) using the following settings: objective = EC plan-Neofluar 40×/1.3 oil; wavelength of excitation 405 nm for DAPI and 488 nm for TUNEL staining. We obtained 25 tiled images (sample in five-frame stack with 3.5-μm interval). The stacks were converted to orthogonal projections and the osteoblasts were counted using Zen 2.3 software (Zeiss, Inc.).
Palmieri M., Kim H., Gomez‐Acevedo H., Que X., Tsimikas S., Jilka R.L., Manolagas S.C., Witztum J.L, & Ambrogini E. (2020). A Neutralizing Antibody Targeting Oxidized Phospholipids Promotes Bone Anabolism in Chow‐Fed Young Adult Mice. Journal of Bone and Mineral Research, 36(1), 170-185.
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5

Quantifying Cell Death In Vitro

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Cell death was assessed by cytotoxicity analyzed by measuring the release of LDH into media according to the manufacture’s protocol (Roche, 4744926), and cell viability determined by MTT assay (Roche, 11465007001) in vitro. TUNEL assay was performed using a DeadEnd fluometric TUNEL system (Promega, G3250) according to manufacturer’s instructions. The TUNEL positive cells in lung were detected using fluorescence microscopy (Nikon, Tokyo Japan). The average number of dead cells was assessed by manual counting of TUNEL+ cells in each high power field (×200).
Yoshida M., Minagawa S., Araya J., Sakamoto T., Hara H., Tsubouchi K., Hosaka Y., Ichikawa A., Saito N., Kadota T., Sato N., Kurita Y., Kobayashi K., Ito S., Utsumi H., Wakui H., Numata T., Kaneko Y., Mori S., Asano H., Yamashita M., Odaka M., Morikawa T., Nakayama K., Iwamoto T., Imai H, & Kuwano K. (2019). Involvement of cigarette smoke-induced epithelial cell ferroptosis in COPD pathogenesis. Nature Communications, 10, 3145.
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