Cell apoptosis was detected by TUNLE assay using a One Step TUNEL apoptosis kit (red Tunnelyte™ CY3 fluorescence detection) (C1089, Beyotime, China) according to the product instruction. Briefly, adherent CCA cells were washed with PBS and then fixed in immunostaining fixative solution (P0098, Beyotime, China) followed by permeabilized in immunostaining strong permeable solution (P0097, Beyotime, China). After that, cells were incubated with TUNEL solution for 1 h and sealed with anti-fluorescence quenching sealing tablets. Lastly, cells were photographed on a fluorescence microscope.
P0097
Manufactured by Beyotime
Sourced in China
The P0097 is a precision weighing instrument designed for laboratory applications. It provides accurate measurements with a maximum capacity of 200 grams and a readability of 0.0001 grams. The device features a durable construction and intuitive user interface, making it suitable for a wide range of laboratory tasks that require precise weight measurements.
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Lab products found in correlation
P0098, by Beyotime (3 mentions)
Tunel staining kit, by Beyotime (2 mentions)
One step tunel apoptosis assay kit, by Beyotime (1 mentions)
P6148, by Merck Group (1 mentions)
Beyoclick edu 594 cell proliferation detection kit, by Beyotime (1 mentions)
C1002, by Beyotime (1 mentions)
Sp5ii, by Leica (1 mentions)
Tunel staining solution, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Wuhan Servicebio Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Ts2 fl, by Nikon (1 mentions)
Hoechst dye, by Beyotime (1 mentions)
Af6000, by Leica (1 mentions)
Ab51608, by Abcam (1 mentions)
S0006, by Affinity Biosciences (1 mentions)
Bl105a, by Biosharp (1 mentions)
Bl539a, by Biosharp (1 mentions)
Hoechst, by Beyotime (1 mentions)
10 protocols using p0097
1
Detecting Cell Apoptosis via TUNEL Assay
2
H2O2-Induced Cell Proliferation Assay
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Cell proliferation activity was detected using an EdU-555 cell proliferation detection kit (C0075S, Beyotime). The cells were inoculated into 6-well plates at a density of 2 × 105 cells per well. The cells were then treated with H2O2 at concentrations of 25, 50, 75, 100, 150, 200, and 300 μM for 12 h. Meanwhile, we designed four treatment methods for further exploration: untreated group, 75 μM H2O2 treatment for 12 h, 300 μM H2O2 treatment for 12 h, 75 μM H2O2 treatment for 12 h and then 300 μM H2O2 treatment for 12 h. Two millilitres of 10 μM EdU reagent was added to each well and incubated for 2 h. Cells were fixed at room temperature for 15 min with 2 ml 4% paraformaldehyde. Two millilitres of immunostaining strong permeability solution (P0097, Beyotime) was added to each well and incubated for 15 min. Two millilitres of Click Additive Solution was added to each well and incubated for 30 min at room temperature (shielded from light). The nuclei were stained with Hoechst 33,358 solution (C0021, Solarbio, Beijing, China). Immunofluorescent staining images were captured by a fluorescence microscope (IX73, Olympus, Tokyo, Japan).
3
Quantitative Apoptosis Assessment via TUNEL
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One Step TUNEL Apoptosis Assay Kit from Beyotime Biotechnology (C1088, Shanghai, China) was used to detect cell apoptosis. Our TUNEL assay was strictly conducted according to the manufacturer’s instructions. Briefly, the cell suspension was fixed in 4% paraformaldehyde for 30 minutes and washed with PBS. Then, the cells were resuspended with immunostaining permeabilization solution (P0097, Beyotime Biotechnology, Shanghai, China) and incubated at room temperature for 5 minutes. Then, the TUNEL reagent (including TdT enzyme, fluorescent labeling solution, and TUNEL detection solution) was prepared according to the manual and added to the cells to incubate for an hour. Lastly, the cells were washed with phosphate-buffered saline (PBS) and the fluorescence was observed under the microscope. TUNEL-positive cells were counted under each field.
4
Quantifying Cell Proliferation with EdU Assay
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Proliferation of Ishikawa and RL95‐2 cells was detected by BeyoClick EdU‐594 cell proliferation detection kit (C0071S, Beyotime). After cells (1 × 105 cells/well in a 6‐well plate) were incubated overnight, the abovementioned treatment procedures were conducted. Then, the cell culture medium was replaced with EdU medium (10 μM), and the cells were continually cultivated for 2 h. Next, the cells were harvested and treated by 4% paraformaldehyde (P6148, Sigma‐Aldrich) and immunostaining strong permeabilization solution (P0097, Beyotime), followed by the staining with EdU fluorescent dye and 4′,6‐diamidino‐2‐phenylindole (DAPI; C1002, Beyotime). Finally, the cell proliferation was observed under a XSP‐63XDV fluorescence microscope (200×, Shanghai optical instrument factory).
5
Immunohistochemistry Analysis of Skin Tissues
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Paraffin-embedded skin tissues were sectioned into pieces and then mounted on slides. After permeabilized with the immunostaining permeable fluid (Beyotime, P0097) for 15 min, slides were blocked with the PBST buffer containing 2% BSA for 15 min, and incubated with the corresponding primary antibody overnight at 4 °C and then with the second antibody for 1 h. Finally, slides are sealed with a drop of sealing tablet and imaged on a confocal laser scanning microscope (LEICA-SP5II).
6
Apoptosis Detection via TUNEL Assay
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The level of cell apoptosis was detected using a TUNEL staining kit (Beyotime, China). In brief, after a 48-hour incubation with or without H2O2, the cells were fixed in 4% paraformaldehyde (Servicebio, China) for 30 min and incubated with a permeabilization solution (P0097, Beyotime, China) for 5 min. The cells were then incubated with the TUNEL staining solution (Beyotime, China) for 1 hour. The cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, China) and observed under a fluorescence microscope. For evaluation of the live/dead cell ratio and cell proliferation, a Live/Dead staining and a CCK-8 assay were performed following the procedure described above.
7
Proliferation Assay of Adipose Stromal Cells
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The assay was performed using an EdU kit (C0071, Beyotime Biotech, Shanghai, China). SVF from eWAT were cultured in conditioned medium of Raw264.7 macrophages. Then, cells were incubated with EdU and permeated with strong permeability solution (P0097, Beyotime Biotech, Shanghai, China) for ten minutes. The cells were placed in the reaction mixture and incubated in the dark for 0.5 h and staining with Hoechst 33342 (C1022, Beyotime Biotech, Shanghai, China). The scale bars for the images shown in figure represents 100 μm. All images were acquired using an optical microscope (TS2FL, Nikon, Tokyo, Japan). Cell numbers were calculated using automated particle analysis in ImageJ (National Institutes of Health, Bethesda, ML, USA).
8
Quantifying DNA Damage via Gamma-H2AX
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Pre-treated cells were washed with phosphate-buffered saline and fixed with fixation solution (P0098, Beyotime, China) for 20 min, followed by permeabilization with permeabilization solution (P0097, Beyotime, China) for 15 min. After blocking with 3% bovine serum albumin (BSA) for 30 min, cells were incubated overnight with gammaH2AX antibody (1:400, 9718, CST, USA), followed by incubation with donkey anti-rabbit fluorescent secondary antibody (1:500, 4412, CST, USA) for 1 h the next day. Nuclei were counterstained with Hoechst dye (1:1000, C1011, Beyotime, China) for 15 min, and fluorescence analysis was performed using a fluorescence microscope (BX63, Olympus, Japan).
9
Immunofluorescence Staining of HIF-1α in Cells
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The cells were fixed with 4% formaldehyde (BL539A, Biosharp, Beijing, China) for 20 min and then incubated with 0.1% Triton X-100 (P0097, Beyotime Biotechnology, Shanghai, China) at room temperature for 20 min. After being blocked with 5% BSA for 1 h at room temperature, the cells were incubated with human anti-HIF-1α antibody (1:500, ab51608, Abcam, Cambridge, UK) at 4 °C overnight. After being washed 3 times with PBS, the cells were incubated with Goat Anti-Rabbit IgG(H + L) Fluor 594-conjugated (1:200, S0006, Affinity Biosciences, Cincinnati, OH, USA) in the dark for 1 h at room temperature. Finally, the nucleus was stained with stained with DAPI (BL105A, Biosharp, Beijing, China) for 5 min at room temperature. After being washed 3 times with PBS, the cells were observed under a fluorescence microscope (AF6000, Leica, Wetzlar, Germany).
10
TUNEL Assay for Apoptosis Analysis
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TUNEL staining kit (Beyotime Biotech) was used the following protocols. Wash NP cells with PBS and fix them for 30 min with fixation solution (P0098, Beyotime Biotech). Wash again with PBS. Put cells in strong permeable solution (P0097, Beyotime Biotech) and incubate for 5 min at room temperature. Prepare TUNEL detection solution: 25 μL TdT enzyme +225 μL fluorescent labeling solution +250 μL TUNEL detection solution. Add 50 μL TUNEL detection solutions to the samples. Put the samples away from light for incubation at 37°C for 60 min. Wash with PBS for 3 times, then add Hoechst (C1017, Beyotime Biotech) for staining for 10 min. Wash with PBS for 3 times. Anti-fluorescence quenching solution was used to seal the slides before observation.
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