Cell apoptosis was measured by detection of active caspase‐3/7 bioluminescence, assayed in the stored tissue homogenates with Caspase‐Glo® 3/7 Assay (Promega, Madison, Wisconsin, USA)14 . The log(Tend/T0) values for WHCO2 and control groups were compared. A second marker of cell viability was determined using the DeadEnd™ Fluorometric TUNEL System (Promega). The extent of DNA fragmentation (a surrogate marker for apoptosis) was quantified by the measurement of green fluorescence intensity with fluorescence microscopy. A counterstain using DAPI (4′‐6‐diamidino‐2‐phenylindole) was performed and detected as blue fluorescence. Images were captured using Zeiss Axio AX10 light microscopy (Carl Zeiss, Oberkochen, Germany) with fluorescence camera AxioCam ICm 1 (Carl Zeiss). TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labelling)‐stained images were reviewed by five scientists/pathologists independently, who were all blinded to sample identity, and the results analysed.
Ax10 axio
Manufactured by Zeiss
Sourced in Germany
The Ax10 Axio is a laboratory microscope designed for optical imaging and analysis. It features high-quality optics and a robust construction to provide reliable performance in various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam icm1, by Zeiss (1 mentions)
Caspase glo 3 7 assay, by Promega (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Axiocam 105 color, by Zeiss (1 mentions)
E cadherin, by Proteintech (1 mentions)
Sa00013 2, by Proteintech (1 mentions)
Coralite488 conjugated affinipure goat anti rabbit igg, by Proteintech (1 mentions)
Sa00013 3, by Proteintech (1 mentions)
Coralite594 conjugated goat anti mouse igg, by Proteintech (1 mentions)
Coverslip, by NEST Biotechnology (1 mentions)
12 well plate, by NEST Biotechnology (1 mentions)
Jc 1 mitochondrial membrane potential detection kit, by Beyotime (1 mentions)
7 protocols using ax10 axio
1
Apoptosis Quantification in WHCO2 Cells
2
Histological Assessment of Peritoneal Architecture
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Peritoneal architecture was examined by light microscopy after staining with haematoxylin and eosin. This approach has been used previously to assess peritoneal effacement20 , 21 , 22 , 23 , 24 . Images were obtained using a Zeiss Axio AX10 light microscope (Carl Zeiss) with a digital camera output (Zeiss Axiocam 105 color; Carl Zeiss). The stained images were assessed independently by five pathologists at the University of Sydney Medical School, all blinded to sample assignment; their results were combined, analysed and quantified.
3
EdU Assay for Cell Proliferation
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The EdU assay was performed to determine cell proliferation. HK-2 cells were seeded onto cover slips in 12-well plate at the density of 50,000 cells/well, and treated with cisplatin (2 μg/mL) in the presence or absence of raloxifene (1 μM or 10 μM) for 24 h. After rinsing in PBS, the cell line was incubated with 1 μM EdU at 37°C for 2 h. Following incubation, the cell line was fixed with 4% paraformaldehyde for 15 min, incubated with 0.5% Triton X-100 for 15 min, and washed 3 times with PBS. Subsequently, the cell line was incubated with a 0.5 mL click additive solution at ambient temperature for 30 min in the dark. After counterstaining with DAPI at 37°C for 10 min, the cells were observed under a fluorescence microscope (Zeiss, Ax10 Axio, Germany).
4
Evaluating Cellular Responses to Res Treatment
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Hematoxylin-Eosin (H/E) staining was performed on cell-bearing coverslips to observe the morphological changes of Res-treated THJ-16T, THJ-21T and Nthy-ori 3-1 cells by the method described elsewhere 34 . 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay, EdU staining were performed to elucidate proliferative activity and trypan blue cell discrimination assay for cell death rates. Deoxynucleotidyl transferase-mediated dUTP-biotin nick and labeling assay (TUNEL, Beyotime Biotechnology, C1086, Shanghai, China) was used to analyze apoptotic cell death by the methods described previously 33 (link). The cell images were collected under a positive fluorescence microscope (Zeiss, Ax10 Axio, Germany).
5
Proliferation and Apoptosis Visualized
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EdU fluorescent labeling was performed to elucidate proliferative activity of THJ-16T/R cells and organoids. Deoxynucleotidyl transferase-mediated dUTP-biotin nick and labeling assay (TUNEL, Beyotime Biotechnology, C1086) was used to analyze apoptotic cell death. Briefly, the cell-bearing coverslips and the organoids of each of the experimental groups were labeled with 5-ethynyl-2'-deoxyuridine (EdU) for 2 h, followed by 30 min incubation with click additive solution at room temperature in darkness. For apoptotic assay, the cell and organoid samples were incubated with TUNEL reaction solution at 37 °C for 60 min, and the cell nuclei were stained with Hoechst in dark for 10 min. The cell images were collected under a positive fluorescence microscope (Zeiss, Ax10 Axio, Germany).
6
Immunocytochemical and Immunofluorescent Staining of NIS and PTEN
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Immunocytochemical staining/ICC was performed on the cell-bearing coverslips obtained from each of the experimental groups by the method described elsewhere 31 (link). The antibodies used were: NIS (Bioss, bs-0448R, Beijing, China; 1:500), PTEN (Wanleibio, WL01901, Shenyang, China; 1:250), E-cadherin (Proteintech, 20874-1-AP, Chicago, USA; 1:500). The color reaction was performed by using 3,30-diaminobenzidine tetrahydrochloride (DAB). For double immunofluorescent staining/IF, after washed with PBS 3 times and then blocked by normal goat serum for 30 minutes at 37°C, the cell-bearing coverslips were co-incubated with rabbit anti-NIS (1:250) and mouse anti-PTEN (Bioss, bsm-33320M, Beijing, China; 1:250) overnight at 4°C in a humid chamber. Then, the coverslips were co-incubated with coralite488-conjugated affinipure goat anti-rabbit IgG (1:500, Proteintech, SA00013-2, Chicago, USA) and coralite594-conjugated goat anti-mouse IgG (1:500, Proteintech, SA00013-3, Chicago, USA) at 37°C for 60 minutes in the dark. Cell nucleus were stained with Hoechst, sealed with fluorescence mounting medium, and observed and imaged under a positive fluorescence microscope (Zeiss, Ax10 Axio, Germany).
7
Assessing Mitochondrial Membrane Potential
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HK-2 cells were seeded onto cover slips (NEST Biotechnology, China) in a 12-well plate (NEST Biotechnology, China) at the density of 50,000 cells/well, and treated with cisplatin with or without raloxifene as described for 24 h. The cells were fixed with 4% paraformaldehyde and stained with 5 mg/L JC-1 (Mitochondrial Membrane Potential Detection Kit) (Beyotime Biotechnology, China) at 37°C for 1 h. After counterstaining with DAPI at 37°C for 10 min, the cells were observed under a fluorescence microscope (Zeiss, Ax10 Axio, Germany). JC-1 accumulates in the mitochondrial matrix in a red-fluorescing polymeric form at high membrane potential and as a monomer emitting green fluorescence signals at low mitochondrial membrane potential. The ratio of green/red JC-1 fluorescence was evaluated to assess membrane depolarization.
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