The EDU assay was carried out using a BeyoClick™ EdU-488 Proliferation Detection Kit (Beyotime, Suzhou, China). In brief, while PC cells adherence in 6-well plate, primary medium was removed and fresh medium were added. Then, total 10μM EDU was injected into each well and cells were cultured in 37°C for 2.5h. Following by xation using 4% paraformaldehyde (Beyotime, Suzhou, China) for 15 min and permeabilization using 0.3% Triton X-100 (Boster, Wuhan, China) for 8 min, total 500μl Apollo dyeing reaction buffer was then added for 40 min in dark environment. After staining the nuclear using DAPI for 10 min, the proportion of EDU was detected using uorescence microscopy.
Triton x 100
Manufactured by Boster Bio
Sourced in China
Triton X-100 is a non-ionic detergent commonly used in various laboratory applications. It functions as a solubilizing agent, capable of disrupting cell membranes and extracting proteins from biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Beyoclick edu 488 proliferation detection kit, by Beyotime (5 mentions)
Paraformaldehyde (pfa), by Beyotime (5 mentions)
Edu reagent, by RiboBio (2 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Boster Bio (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Beyotime (1 mentions)
11 protocols using triton x 100
1
Cell Proliferation Assay Using EDU
2
Cell Proliferation Assay Using EDU
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The EDU assay was carried out using a BeyoClick™ EdU-488 Proliferation Detection Kit (Beyotime, Suzhou, China). In brief, while PC cells adherence in 6-well plate, primary medium was removed and fresh medium were added. Then, total 10μM EDU was injected into each well and cells were cultured in 37°C for 2.5h. Following by xation using 4% paraformaldehyde (Beyotime, Suzhou, China) for 15 min and permeabilization using 0.3% Triton X-100 (Boster, Wuhan, China) for 8 min, total 500μl Apollo dyeing reaction buffer was then added for 40 min in dark environment. After staining the nuclear using DAPI for 10 min, the proportion of EDU was detected using uorescence microscopy.
3
Cell Proliferation Assay Using EDU
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The EDU assay was carried out using a BeyoClick™ EdU-488 Proliferation Detection Kit (Beyotime, Suzhou, China). In brief, while PC cells adherence in 6-well plate, primary medium was removed and fresh medium were added. Then, total 10μM EDU was injected into each well and cells were cultured in 37°C for 2.5h. Following by xation using 4% paraformaldehyde (Beyotime, Suzhou, China) for 15 min and permeabilization using 0.3% Triton X-100 (Boster, Wuhan, China) for 8 min, total 500μl Apollo dyeing reaction buffer was then added for 40 min in dark environment. After staining the nuclear using DAPI for 10 min, the proportion of EDU was detected using uorescence microscopy.
4
Cell Proliferation Assay Using EDU
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The EDU assay was carried out using a BeyoClick™ EdU-488 Proliferation Detection Kit (Beyotime, Suzhou, China). In brief, while PC cells adherence in 6-well plate, primary medium was removed and fresh medium were added. Then, total 10μM EDU was injected into each well and cells were cultured in 37°C for 2.5h. Following by xation using 4% paraformaldehyde (Beyotime, Suzhou, China) for 15 min and permeabilization using 0.3% Triton X-100 (Boster, Wuhan, China) for 8 min, total 500μl Apollo dyeing reaction buffer was then added for 40 min in dark environment. After staining the nuclear using DAPI for 10 min, the proportion of EDU was detected using uorescence microscopy.
5
EdU Incorporation Assay for Cell Proliferation
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EdU reagent was purchased from Ribobio (Guangzhou, China). Transfected cells were cultured in confocal dishes and then washed and fixed after 24 h. Next, 0.2% Triton X-100 (Boster, Wuhan, China) was used to treat cells for 10 min. Cells were then incubated with the EdU reagent for 25 min and stained with Hochest33258 for 10 min. Images were captured by a fluorescence microscope.
6
EdU Incorporation Assay Protocol
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The EdU reagent was purchased from RiboBio (Guangzhou, China). The transfected cells were cultured in confocal dishes, washed, and fixed for 24 h. Next, cells were treated with 0.2% Triton X-100 (Boster, Wuhan, China) for 10 min, incubated with the EdU dye agent for 25 min, and stained with DAPI for 10 min. Finally, images were captured using a fluorescence microscope.
7
Visualizing SAPK/JNK Phosphorylation
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After fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.2% TritonX-100 (Boster Biological Technology) for 5 min, the cells were blocked in 5% bovine serum albumin (BSA, diluted in PBS) for 30 min. The cells were then incubated overnight at 4 °C with primary antibodies. Primary rabbit polyclonal antibody Phospho-SAPK/JNK (Thr183/Tyr185) (#9255, CST, MA, USA) was diluted 1:50 in blocking buffer. Fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Proteintech, Chicago, USA) was diluted 1:200 in blocking buffer and applied to cells at RT in the dark for 1 h, followed by stained with 4′, 6-diamidino-2-phenylindole (DAPI, 1:1000) at RT in the dark for 10 min. The confocal fluorescence microscopy (PerkinElmer, Waltham, Massachusetts, USA) was used to capture the immunofluorescence images.
8
Quantifying Cell Proliferation with EDU Assay
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The EDU assay was carried out using a BeyoClick™ EdU-488 Proliferation Detection Kit (Beyotime, Suzhou, China). In brief, PC cells were cultured in 6-well plates and were allowed to adhere. The primary culture medium was removed and fresh medium was added. Then, 10μM EDU was added into each well and cells were cultured in 37°C for 2.5h. After that, cells were fixed in 4% paraformaldehyde (Beyotime, Suzhou, China) for 15 min and permeabilization using 0.3% Triton X-100 (Boster, Wuhan, China) for 8 min. Then, 500μl Apollo dyeing reaction buffer was added for 40 min in the dark. After staining, the nuclei were stained using DAPI for 10 min. The EDU staining was observed under a fluorescence microscope (Zeiss, Oberkochen, Germany).
9
Immunofluorescence Staining of PC Cells
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First, PC cells were cultured on slides overnight, then fixed with 4% paraformaldehyde for 30 min and infiltrated with 0.1% Triton X-100 (Boster Biological Technology) for 5 min. Later, 5% BSA was added as a blocking buffer and incubated for 1 h. The cells were then incubated with different primary antibodies at 4 °C overnight. CY3-conjugated goat anti-rabbit antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody were diluted at 1:200 in blocking buffer and treated at 37 °C for 1 h, followed by DAPI staining for 5 min. Lastly, images were obtained by a laser scanning confocal microscope.
10
Immunofluorescence Assay for Citrullinated Proteins
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The immunofluorescence assays for peptidyl citrulline, histone H3 peptidyl citrulline, PAD2, PAD4 and specificity protein-3 (SP3) were performed just as previously described30 (link). The RAW264.7 or neutrophil-like cells were fixed in methyl alcohol, permeabilized with 0.1% Triton X-100 (Boster Biological Technology, Wuhan, China) for 20 min and blocked with 3% BSA (Boster Biological Technology, Wuhan, China) for 1 h. Next, the cells were incubated with peptidyl citrulline, histone H3 peptidyl citrulline, PAD2, PAD4 and SP3 antibodies overnight at 4 °C, and then with Alexa Fluor 594 or Alexa Fluor 488 conjugated IgG for 1 h at 37 °C. The cells were counter-stained with DAPI (Solarbio Life Science, Beijing, China) for 15 min, and photographed using a fluorescence microscope (Olympus, Tokyo, Japan) and the mean fluorescence intensity of each sample was analyzed by ImageJ (version: 1.53q, NIH, USA).
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