RGCs were treated or transfected and then cultured 48 h. After rising twice with PBS, the cells were fixed with 4% paraformaldehyde for 15 min at 37 °C. 1×104 cells were prepared in 96-well plates and then subjected to Click-iTTM Plus TUNEL Assay Kit (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Following DAPI staining, the apoptotic cells were measured by using Nikon Eclipse 80i microscope (Nikon Corporation).
Click ittm plus tunel assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Click-iT™ Plus TUNEL Assay Kit is a tool for detecting and quantifying apoptosis, a type of programmed cell death. It utilizes a specific labeling reaction to identify DNA fragmentation, a hallmark of apoptosis. The kit provides a straightforward method to visualize and analyze apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse 80i microscope, by Nikon (3 mentions)
Vectashield plus dapi, by Vector Laboratories (1 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
Dapi solution, by Vector Laboratories (1 mentions)
Mouse anti ctnt, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc apop dtec kit, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
6 protocols using click ittm plus tunel assay kit
1
Click-iT TUNEL Assay for Apoptosis
2
Quantifying Apoptosis in SGC Cells
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SGC cells were treated or transfected and then cultured for 48 hours with a 2 PBS wash. After this, SGC cells underwent 4% PFA fixation for 15 minutes at 37 ℃. 2×104 cells were prepared in 96-well plates and then subjected to Click-iTTM Plus TUNEL Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Following DAPI staining, the apoptotic cells were measured by using a Nikon Eclipse 80i microscope (Nikon Corporation, Japan).
3
Quantifying Apoptosis in RGC-5 Cells
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RGC-5 cells were treated or transfected and then cultured for 48 hours with a 2 PBS wash. After this, RGC-5 cells underwent 4% paraformaldehyde fixation for 15 minutes at 37 ℃. 1×104 cells were prepared in 96-well plates and then subjected to Click-iTTM Plus TUNEL Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Following DAPI staining, the apoptotic cells were measured by using a Nikon Eclipse 80i microscope (Nikon Corporation, Japan).
4
Paraffin Tissue TUNEL Assay Protocol
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Paraffin-embedded tissues were cut on a Reichert-Jung 2030 microtome into 10 μm sections and placed on Thermo Scientific Superfrost Plus microscope slides. Sections were adhered onto slides in an oven at 55–60 °C for 1 h. Samples were deparaffinized and stained using a Click-iTTM Plus TUNEL Assay Kit (Thermo Fisher C10618) following the manufacturer’s instructions. Slides were washed 3X with PBS and mounted with Vectashield plus DAPI (Vector Laboratories).
5
Quantifying Cardiomyocyte Apoptosis via TUNEL
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TUNEL staining was applied using a Click-iTTM Plus TUNEL assay kit
(Invitrogen) according to the manufacturer’s instruction. Heart tissues were
hydrated, fixed in 4% Paraformaldehyde (Chemcruz) and permeabilized with
Proteinase K. Samples were then fixed again with 4% PFA and incubated with TUNEL
reaction cocktail. Finally, samples were stained with a primary antibody
specific for cardiac troponin T (1:100; mouse anti-cTnT; Thermofisher) and
secondary antibody (1:500; Alexa 488 anti-mouse; Thermofisher) mounted with DAPI
solution (Vector laboratory). Samples were imaged under a confocal microscope
using a 20X objective. Five views were randomly selected within the infarcted
zone from each section to assess the ratio of apoptotic CMs.
(Invitrogen) according to the manufacturer’s instruction. Heart tissues were
hydrated, fixed in 4% Paraformaldehyde (Chemcruz) and permeabilized with
Proteinase K. Samples were then fixed again with 4% PFA and incubated with TUNEL
reaction cocktail. Finally, samples were stained with a primary antibody
specific for cardiac troponin T (1:100; mouse anti-cTnT; Thermofisher) and
secondary antibody (1:500; Alexa 488 anti-mouse; Thermofisher) mounted with DAPI
solution (Vector laboratory). Samples were imaged under a confocal microscope
using a 20X objective. Five views were randomly selected within the infarcted
zone from each section to assess the ratio of apoptotic CMs.
6
Apoptosis Assessment by Flow Cytometry
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Apoptosis was assessed by ow cytometric analysis and terminal deoxynucleotidyl trans-ferasemediated dUTP nick end labeling (TUNEL). TUNEL assays were performed using the Click-iT TM Plus TUNEL assay kit (Invitrogen, USA) according to the manufacturer's instructions. Flow cytometric analysis was performed using the Annexin V FITC Apop Dtec Kit (BD Pharmingen, USA) according to the manufacturer's instructions. The cells were trypsinized and centrifuged at 300 × g for 10 min at 4°C. Afterward, the cell pellets were resuspended in binding buffer and stained with Annexin V FITC and propidium iodide for 15 min on ice. Apoptotic cells were analyzed by ow cytometry (BD FACS Calibur, USA).
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