SW620 cells were seeded in 6-well plates and then treated with vehicle, IL-6, and/or α-hederin, or PDTC at different concentrations for 24 h. Apoptosis was evaluated using a Hoechst 33258 staining kit provided by Beyotime Institute of Biotechnology (Haimen, China) according to the protocol. Morphology of apoptotic cells was observed using a fluorescence microscope (Nikon, Tokyo, Japan). The nucleus of apoptotic cells takes up the Hoechst reagent and exhibits a bright blue fluorescence. Results were from experiments in triplicate.
Hoechst 33258 staining kit
Manufactured by Beyotime
Sourced in China
Hoechst 33258 is a fluorescent dye used for staining DNA in biological samples. It binds to the minor groove of double-stranded DNA, allowing visualization of nucleic acid content in cells or tissues. The Hoechst 33258 staining kit provides the necessary reagents and protocol to perform this staining procedure.
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Lab products found in correlation
Trypsin, by Thermo Fisher Scientific (4 mentions)
Bx63 microscope, by Olympus (3 mentions)
Temozolomide, by Thermo Fisher Scientific (3 mentions)
Temozolomide, by Selleck Chemicals (3 mentions)
Fluorescence microscope, by Nikon (2 mentions)
N acetylcysteine nac, by Merck Group (2 mentions)
Caspase 3 colorimetric assay kit, by Abcam (2 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Reactive oxygen species assay kit, by Beyotime (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mpeg plga, by Jinan Daigang Biomaterial (1 mentions)
Flow cytometry, by Beckman Coulter (1 mentions)
Elisa microplate reader, by Bio-Rad (1 mentions)
Rabbit anti ampk, by Cell Signaling Technology (1 mentions)
Rabbit anti bax, by Cell Signaling Technology (1 mentions)
Cisplatin, by Merck Group (1 mentions)
45 protocols using hoechst 33258 staining kit
1
Apoptosis Evaluation of SW620 Cells
2
Hoechst 33258 Staining of Apoptotic GC Cells
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Hoechst 33258 Staining Kit (Beyotime, Shanghai, China) was used to detect the morphological features of apoptotic cells. Exponentially growing GC cells were placed into sterile 6-well plates at 1×105 cells/well and incubated for 24 h. Subsequently, the cells in the wells were treated with hesperetin (200 μM), DDP (4 μg/ml), or hesperetin + DDP (200 μM + 4 μg/ml) for another 24 h. Ultimately, the cells were stained with Hoechst 33258 according to the manufactures’ instructions. Additionally, transfected GC cells were also exposed to hesperetin + DDP (200 μM + 4 μg/ml) for 24 h and stained as described above. The morphological features of the apoptotic cells were observed under a fluorescent microscope (BX51, Olympus, Tokyo, Japan). The ratio of the apoptotic cell number to the total cell number defined the apoptosis ratio.
3
Apoptosis Detection via Hoechst 33258 Staining
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Hoechst 33258 Staining Kit (Beyotime) was used to detect the apoptotic morphological features. Cells in exponential growth was seeded into a six-well plate (1×105 cells/well). GC cells were cultured into a six-well plate for 24h, and treated with TQ and cisplatin as mentioned above, and further stained with Hoechst 33258. Additionally, transfected GC cells were also exposed to the combination of TQ and cisplatin for 24h and stained as described above. Apoptotic morphological features was observed and captured using a fluorescent microscope (BX51, Olympus).
4
Apoptosis Analysis by Hoechst 33258 Staining
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Cell apoptosis was examined by employing Hoechst 33258 Staining Kit (Beyotime). HGC27/DDP cells were cultured into a 6-well plate (1×105cells/well) with fresh medium for 24 hrs and then treated with α-hederin for 0, 5 μM, 10 μM, 15 μM and 10 μM with or without pretreatment 8 mM DL-buthionine-S, R-sulfoximine (BSO, Sigma-Aldrich) or 12 mM N-acetylcysteine (NAC, Sigma-Aldrich) for 2 hrs for another 24 hrs. Subsequently, the cells were fixed by 4% paraformaldehyde solution for 20 mins, washed twice with PBS and stained with Hoechst 33258 for 20 mins at room temperature in the dark. A fluorescence microscope (BX51, Olympus, Japan) was applied to observe and capture the apoptotic morphological features such as chromatin condensation and nuclear fragmentation.
5
Rhynchophylline Compound Characterization
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Rhynchophylline (purity>98%) was purchased from Beisite Biotechnology Co., Ltd. (Chengdu, China). For in vitro cell studies, rhynchophylline was dissolved in dimethyl sulfoxide (DMSO) and further diluted in culture medium (DMEM with 10% fetal bovine serum) to the required concentration with the final concentration of DMSO concentration <0.1% (v/v). Cell culture plates and transwell plates were obtained from Corning INC. (Corning, NY, USA). Hoechst 33258 staining kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). mPEG-PLGA (MW = 15 kDa; LA/GA = 75:25; PEG MW = 2 kDa) was purchased from Jinan Daigang Biomaterial Co., Ltd. (Jinan, China). Poly(vinyl alcohol) (PVA, MW=30–70 kDa, HD, 80%), 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and Tween 80 were purchased from Chengdu Real Biotechnology Co., Ltd. All other reagents were of analytical grade and were used as supplied.
6
Apoptosis Assessment Techniques
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Morphological assessment of apoptotic cells was performed using Hoechst 33258 staining kit (Beyotime, Shanghai, China) according to the supplier's protocols. Briefly, the cells were fixed in 4% paraformaldehyde for 10 min and were washed twice with PBS. Then, the cells were stained with 0.5 ml of Hoechst 33258 staining for 5 min and were observed under a fluorescence microscope at 350 nm.
The annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD, San Jose, CA, USA) was used to assess cell apoptosis according to the supplier's protocols. Forty-eight hours after transfection, cells were collected, counted, centrifuged, and resuspended to 5 × 105 cells in 500 μl of 1 × binding buffer. Annexin V-FITC (5 μl) and 10 μl PI were added to each tube. The samples were incubated in the dark at room temperature for 15 min. Samples were then examined immediately on a flow cytometry (Beckman, Fullerton, CA, USA).
Cell apoptosis was examined by analyzing the activity of Caspase-3 using the Enzyme-linked immunosorbent assay kit (R and D, Minneapolis, MN, USA) according to the manufacturer's instructions. The optical density (OD) values were measured using an ELISA microplate reader (Bio-Rad, Hercules, CA, USA).
The annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD, San Jose, CA, USA) was used to assess cell apoptosis according to the supplier's protocols. Forty-eight hours after transfection, cells were collected, counted, centrifuged, and resuspended to 5 × 105 cells in 500 μl of 1 × binding buffer. Annexin V-FITC (5 μl) and 10 μl PI were added to each tube. The samples were incubated in the dark at room temperature for 15 min. Samples were then examined immediately on a flow cytometry (Beckman, Fullerton, CA, USA).
Cell apoptosis was examined by analyzing the activity of Caspase-3 using the Enzyme-linked immunosorbent assay kit (R and D, Minneapolis, MN, USA) according to the manufacturer's instructions. The optical density (OD) values were measured using an ELISA microplate reader (Bio-Rad, Hercules, CA, USA).
7
Hoechst 33258 Staining for Neuronal Apoptosis
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Hoechst 33258 staining kit (Beyotime Biotechnology Inc.) was used to detect neuronal apoptosis [53 ]. After Hoechst staining, the nucleus of normal neurons appeared blue, while the nucleus of apoptotic neurons appeared dense or fragmented with white color. Neurons were fixed with fixative solution for 20 min and washed with PBS three times, followed by staining with dye solution at room temperature for 5 min. After washing with PBS three times, neurons were sealed with anti-fluorescence quenching sealing solution, and observed under fluorescence microscope.
8
Ginseng Compound G‐Rb3 Protects Against Cisplatin‐Induced Toxicity
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G‐Rb3 (purity ≥ 98.5%, HPLC method) was isolated and purified from the leaves of Panax quinquefolium (American ginseng). Cisplatin was purchased from Sigma Chemicals with purity more than 99%. Compound C, rapamycin (Ram) and acetylcysteine (NAC) also were purchased from MedChemExpress Biotech and stored at −80°C in darkness. The commercial assay kits for determining reduced glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), blood urea nitrogen (BUN) and creatinine (CRE) were bought from Nanjing Jiancheng Biological Research Institute. Haematoxylin and eosin (H&E) dying kit and Hoechst 33258 staining kit were obtained from Beyotime Co, Ltd. The immunohistochemically assay kits together with SABC‐DyLight488 immunofluorescence staining kits were obtained from BOSTER Biological Technology Co, Ltd. The primary rabbit monoclonal antibodies including anti‐LC3, anti‐BNIP3, anti‐β‐actin, anti‐GAPDH, anti‐Atg3, anti‐Atg5, anti‐Atg7 and anti‐p62 were all provided by BOSTER Biological Technology Co, Ltd. The rabbit anti‐AMPK, rabbit anti‐mTOR, rabbit anti‐Bax, Bcl‐2, Bad, caspase 3 and caspase 9 were acquired from Cell Signaling Technology. TUNEL commercial kit was purchased from Roche Applied Science. All other reagents and chemicals, unless indicated, were obtained from Beijing Chemical Factory.
9
Apoptosis Measurement in Bladder Cancer
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Cell apoptosis was revealed by both Hoechst 33258 staining assay and ELISA assay. Apoptosis ratio in bladder cancer cells was measured using Hoechst 33258 staining kit (Beyotime, Shanghai, China), and Caspase-3 activity was measured using a Caspase-3 Colorimetric Assay kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions at 48 hours after transfection. Experiments were repeated at least three times in duplicates.
10
Hoechst 33258 Apoptosis Assay
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Apoptosis was evaluated using a Hoechst 33258 staining kit (Beyotime Biotechnology). The cells were harvested, washed, mixed with 4% (v/v) paraformaldehyde at 25°C for 30 min, further washed, and stained with 500 μL of Hoechst 33258 for 5 min. Fluorescence microscopy (LI-COR, Lincoln, NE, USA) was used to observe nuclear morphology.
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