Transfected KYSE450 and ECA109 cells (2 × 103 cells/well) were seeded into 96‐well plastic dishes and grown under standard conditions for 0–72 h. In the CCK‐8 assay, at 0, 24, 48 and 72 h post‐seeding, we evaluated cell growth using CCK‐8 solution following the manufacturer's recommendations (Yesen). Absorption at 450 nm was determined by spectrophotometry (TECAN). In the EDU assay, at 72 h after cell seeding, we treated the cells with Cell‐Light EdU Apollo488 Kit (Ribobio) and 4′,6‐diamidino‐2‐phenylindole (DAPI, Yesen) (nuclear staining) as previously described.34 The proportion of EDU‐positive cells was scored using a fluorescence microscope (Keyence).
4 6 diamidino 2 phenylindole dapi
Manufactured by Yesen
Sourced in China
4',6-diamidino-2-phenylindole (DAPI) is a fluorescent dye that binds strongly to adenine-thymine (A-T) rich regions in DNA. It is commonly used in various biological and research applications for staining and visualizing nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
A1r eclipse ti, by Nikon (2 mentions)
Cck 8 solution, by Yesen (1 mentions)
Cell light edu apollo 488 kit, by RiboBio (1 mentions)
Fluorescence microscope, by Keyence (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Ab1416, by Abcam (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Ab92547, by Abcam (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Nuclear extraction kit, by Solarbio (1 mentions)
Fitc labeled anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
5 protocols using 4 6 diamidino 2 phenylindole dapi
1
Assessing Proliferation in Esophageal Cancer
2
Detecting Nuclear Translocation of Transcription Factors
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Immunofluorescence staining was performed to detect the nuclear translocation of transcriptional factors, including NF-κB, AP-1, and IRF3, as our previously described.19 (link) Briefly, the cells (1×104) were seeded into a chamber slide for 24 h. Then, the cells were treated with SC (200 and 400 μg/mL) for 1 h. After LPS (1 μg/mL) treatment for 30 min, the cells were fixed with formaldehyde in PBS (w:v, 4%) for 15 min. Subsequently, cells were permeabilized by Triton X-100 (0.25%) for 30 min at 37°C, and then blocked for 1 h with BSA (2%) and incubated with indicated primary antibodies overnight at 4°C. The cells were incubated with fluorescence-labeled goat anti-rabbit secondary antibody at room temperature for 1 h, and washed by PBST for three times. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI, YESEN, Shanghai, China). The slides were mounted by anti-fluorescence quenchers and then photographed under an A1R confocal microscope (Nikon, Japan). For fluorescence intensity quantification, 50 cells were analyzed in each group by ImageJ software as following: images were split into two channels and the nuclei were selected as regions of interest (ROIs). By the ROI Manager, the mean intensity of green fluorescence in the nuclear area was then measured.
3
Immunostaining of Vimentin and E-cadherin in Cells
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Stable transfected cells were seeded onto cover glasses in 6-well plates. When the cells on each glass reached 20-30% confluence, the cells were fixed with 4.0% paraformaldehyde, permeabilized with 0.2% Triton X-100 (Beyotime, Shanghai, China) for 10 min at room temperature, and washed three times with PBS. Finally, the cells were incubated with rabbit anti-human vimentin (ab92547, 1:100, Abcam) or mouse anti-human E-cadherin (ab1416, 1:100, Abcam) antibodies at 4°C overnight. Then, the cells were washed with PBS and incubated with an immunofluorescent-labelled secondary antibody (Yesen, Shanghai, China) in darkness at room temperature for 2 h, followed by three washes (5-10 min each). The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Yesen, Shanghai, China) for 10 minutes. The slides were visualized, and photos were taken with a fluorescence microscope (Olympus, Tokyo, Japan).
4
Visualizing Nuclear Translocation of Transcription Factors
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The nuclear localization of p65, c-Jun, and IRF3 was detected with Nikon A1R Eclipse Ti confocal microscope (Nikon Corp., Tokyo, Japan) as previously described [21] . Brie y, cells were seeded into chamber slide for 12 h, and then treated with FSI (100 and 200 μg/mL) for 1h. The cells were xed by 4% paraformaldehyde for 10 min and then permeabilized with Triton X-100 (0.25%) during 30 min at 37 °C. Cells were then blocked for 1 h with BSA (2%) and incubated with target antibodies including p65, c-Jun and IRF3 diluted in cold PBS containing 3 % BSA at 4°C for overnight. Then, the cells were incubated with 1:1,000 dilution of FITC-labeled anti-rabbit secondary antibody in dark under room temperature for 1 h. After incubating, the cells were washed for 3 times by cold PBS. 4′,6-diamidino-2-phenylindole (DAPI, YESEN, Shanghai, China) was used to stain nucleus right before performing assay.
Cytosolic and nuclear protein separation RAW264.7 cells were seeded (5×10 5 cells/well) into 60 mm-diameter culture dishes and cultured for 24 h. After treatment with FSI for 1 h and followed by LPS stimulation for 30 min, PBS was used to wash cultures, and then using nuclear extraction kit (Solarbio, Beijing, China) to separate nuclear and cytosolic protein. After that, the samples were dissolved in lysis buffer for Western blot assay.
Cytosolic and nuclear protein separation RAW264.7 cells were seeded (5×10 5 cells/well) into 60 mm-diameter culture dishes and cultured for 24 h. After treatment with FSI for 1 h and followed by LPS stimulation for 30 min, PBS was used to wash cultures, and then using nuclear extraction kit (Solarbio, Beijing, China) to separate nuclear and cytosolic protein. After that, the samples were dissolved in lysis buffer for Western blot assay.
5
Nuclear Localization of Transcription Factors
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The nuclear localization of p65, c-Jun, and IRF3 was detected with Nikon A1R Eclipse Ti confocal microscope (Nikon Corp., Tokyo, Japan) as previously described[ 24 ]. Briefly, cells were seeded into chamber slides for 12 h and then treated with flavonoids of S. involucrata (100 and 200 μg/mL) for 1 h. After LPS (1 μg/mL) stimulation for 1 h, cells were fixed by 4% paraformaldehyde for 10 min and then permeabilized with Triton X-100 (0.25%) for 30 min at 37 °C. Cells were then blocked for 1 h with 2% bovine serum albumin and incubated with target antibodies including p65, c-Jun, and IRF3 diluted in cold PBS containing 2% bovine serum albumin at 4 °C overnight. Then, the cells were incubated with FITC-labeled anti-rabbit secondary antibody (Cell Signaling Technology, catalog no. 4412; 1:500 dilution) in dark under room temperature for 1 h. After incubation, cells were washed 3 times by cold PBS. 4',6-diamidino-2-phenylindole (DAPI, YESEN, Shanghai, China) was used to stain nucleus right before performing assay.
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