Cell apoptosis was observed by employing the Click-iT™ Plus TUNEL (the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay kit (ThermoFisher Scientific). Cells were grown on coverslips inside a 6-well plate and treated with male and female M. pomifera extracts at 500 µg/mL concentrations. Twenty hours after treatment, the MCF-7 and T47D cells were fixed with 4% formaldehyde. TUNEL assays were performed according to the manufacturer’s instructions; the nuclear fluorescence of cells was detected and analyzed using a LionHeart FX microscope (Agilent, Santa Clara, CA, USA).
Click it plus tunel
Manufactured by Thermo Fisher Scientific
The Click-iT Plus TUNEL assay is a kit that detects apoptosis-induced DNA fragmentation in cells. It utilizes a specially designed terminal deoxynucleotidyl transferase (TdT) reaction to label the DNA breaks with a fluorescent dye, enabling the visualization and quantification of apoptotic cells.
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Lab products found in correlation
Sp8x confocal microscope, by Leica (2 mentions)
Alexafluor conjugated donkey anti rabbit secondary antibody, by Abcam (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Lionheart fx microscope, by Agilent Technologies (1 mentions)
Alexafluor 546 goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
4 protocols using click it plus tunel
1
Apoptosis Assay of M. pomifera Extracts
2
Quantification of TUNEL-Positive Cells in Tumor Samples
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For TUNEL staining, cryopreserved tumor samples were cryosectioned at 10 μM thick and mounted on superfrost microscope slides. Slides were stored at -80°C and stained using the Click-iT Plus TUNEL (ThermoFisher C10617) according to the manufacturer’s instructions. After TUNEL staining, slides were blocked with 5% normal donkey serum in PBS (Gibco, 10010-050) for one hour and incubated overnight at 4°C with rabbit anti-pan-cytokeratin polyclonal antibody (Abcam ab217916 1:400). The following day, slides were washed three times in PBS containing 0.01% Triton X-100 and incubated for 2 hours with AlexaFluor conjugated donkey anti-rabbit secondary antibody (Abcam ab150073 1:1000). After secondary antibody incubation, slides were washed three times with PBS containing 0.01% Triton X-100 and mounted with ProLong™ Gold Antifade Mountant with DAPI (P36931, Thermo Fisher Scientific). Images were acquired using a Leica Sp8x confocal microscope. TUNEL positive/pan-cytokeratin positive cells were quantified in at least eight randomly sampled 10x fields per sample using QuPath 0.2.3.
3
Quantification of Apoptosis in MHV-1 Pneumonia
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Apoptosis was measured in mice with MHV-1 pneumonia using a TUNEL immunofluorescence assay. Lungs were harvested from control and MHV-1 infected mice, formalin-fixed, paraffin-embedded, sectioned and then dual stained with Click-iT® Plus TUNEL (Thermofisher) and 4′,6-diamidino-2-phenylindole (DAPI). To confirm MHV-1 infection, the same sections were blocked with 3% BSA in PBS at 37 °C for 1 hour and an MHV A59 Nsp9 antibody (NBP-21671, Novus Biologicals, Centennial, CO, USA) was applied (1:500 for 1 hour at 37 °C). Finally an Alexa Fluor 546 goat anti-mouse secondary antibody (Thermofisher, A11035) was applied at 1:400 and the slides were incubated for 1 hour at 37 °C. Images were then captured on a Leica SP8 confocal microscope using a 63X 1.4 NA objective and a white light laser at 488 nm. Emission acquisition gating was 510–530 nm (for TUNEL). A 405 nm laser diode was used for DAPI excitation and the emission acquisition gate was set at 420–480 nm. Images were captured using the tile scan function in LAS X software to acquire a total of 9 tiles per tissue slice in a 4-slice Z-stack. The 3-dimensional volume of TUNEL positive cells was counted using LAS X software.
4
Quantification of Apoptosis in Tumor Samples
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For TUNEL staining, cryopreserved tumor samples were cryosectioned at 10 µM thick and mounted on superfrost microscope slides. Slides were stored at -80ºC and stained using the Click-iT Plus TUNEL (ThermoFisher C10617) according to the manufacturer's instructions. After TUNEL staining, slides were blocked with 5% normal donkey serum in PBS (Gibco, 10010-050) for one hour and incubated overnight at 4ºC with rabbit anti-pan-cytokeratin polyclonal antibody (Abcam ab217916 1:400). The following day, slides were washed three times in PBS containing 0.01% Triton X-100 and incubated for 2 hours with AlexaFluor conjugated donkey anti-rabbit secondary antibody (Abcam ab150073 1:1000). After secondary antibody incubation, slides were washed three times with PBS containing 0.01% Triton X-100 and mounted with ProLong™ Gold Antifade Mountant with DAPI (P36931, Thermo Fisher Scientific). Images were acquired using a Leica Sp8x confocal microscope. Quantification of TUNEL positive/ pan-cytokeratin positive cells was done in at least eight randomly sampled 10x fields per sample using QuPath 0.2.3.
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