The GBM primary cells with rs11558961 CC (n=5), CG (n=3) or GG (n=3) genotypes (5×103 per well) were incubated with imatinib at the concentration of 10μg/ml or 50μg/ml for 24 hours. The cells were then fixed with 4% paraform followed by staining with 200 μl Hoechst 33258 reagent (Beyotime, Shanghai, China) in the dark for 30 min. After being washed twice with PBS, cells were immediately photographed under an inversion fluorescence microscope (Olympus IX51, Tokyo, Japan) to determine ratios of apoptotic cells. In CC, CG or GG group, cells were quantified by counting at least 3 samples, 5 independent visual fields/sample, to determine the apoptotic ratio. Data was presented as mean ± SD.
Hoechst 33258 reagent
Manufactured by Beyotime
Sourced in China
Hoechst 33258 is a fluorescent dye that binds to adenine-thymine (A-T) rich regions of DNA. It is commonly used in various laboratory techniques, such as fluorescence microscopy and flow cytometry, to visualize and quantify DNA content.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Affinity Biosciences (1 mentions)
P erk1 2, by Affinity Biosciences (1 mentions)
Hif 1a, by Affinity Biosciences (1 mentions)
Vegfa, by Affinity Biosciences (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Cleaved caspase 9, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
P pi3k, by Affinity Biosciences (1 mentions)
Ix73 microscope, by Olympus (1 mentions)
6 protocols using hoechst 33258 reagent
1
Apoptosis in GBM Cells by Imatinib
2
Comprehensive Cell Signaling Protocol
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These materials using in this study are mentioned in prior study, such as 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMSO 5-FU, RIPA lysis buffer, crystal violet, 4% paraformaldehyde solution, Dulbecco’s modified eagle medium (DMEM), penicillin-streptomycin, Phosphate buffered saline (PBS), Fetal Bovine Serum (FBS), Pierce BCA protein assay kit, and Super Signal™ West Pico Chemiluminescent Substrate kit, TRIzol lysis buffer, and The Prime Script RT reagent Kit and TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) [10 ,14 ]. Hoechst 33258 reagent was from Beyotime Biotechnology (Jiangsu, China). The primary antibodies against Bcl-2, Bax, cleaved-caspase 3, cleaved caspase 9, GAPDH and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). P-R-Ras, P-ERK1/2, P-PI3K, VEGFA, P-AKT, HIF-1A, and P-ERK1/2 antibodies were purchased from Affinity Biosciences (Cincinnati, OH, USA).
3
Hoechst 33258 Apoptosis Detection
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Cell apoptosis was detected by Hoechst 33258 stain analysis. In brief, 2×105 cells per well were washed with phosphate-buffered saline (PBS) twice. Then, 1 mL of Hoechst 33258 reagent (Beyotime, Nantong, China) was added to each well, and the cells were incubated at 37 °C for 30 min in the dark. Next, the Hoechst 33258 reagent was removed, and cells were washed with PBS 3 times (5 min × 3 times). Morphological changes of apoptotic cells were observed under an inverted fluorescence microscope, and images were captured.
4
UA-Induced Apoptosis in T24 Cells
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T24 cells were disseminated into six-well plates with 5 × 104 cells/well and incubated at 37°C with 5% CO2 for 24 hours. The cells were then treated with UA under different conditions for another 24 hours. The cells were washed three times with phosphate-buffered saline and then treated with Hoechst 33258 reagent (10 μg/mL, Beyotime Institute of Biotechnology) in the dark at room temperature for 10 minutes. Cell morphology was observed using a fluorescence microscope with a blue filter.
5
Hoechst 33258 Apoptosis Assay
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Hoechst 33258 staining was used to detecte apoptosis. In brief, 3×105 cells per well were washed with PBS twice. Then, 1 mL of Hoechst 33258 reagent (Beyotime, Nantong, China) was added to each well, and the cells were incubated at 37 °C for 30 min in the dark. Then, the Hoechst 33258 reagent was removed, and cells were washed with PBS for 3 times (5 min × 3 times). Morphological changes of apoptotic cells were observed under an inverted fluorescence microscope, and images were captured.
6
Hoechst 33258 Staining of HeLa Cells
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The Hela cells were seeded in six-well plates. After treatment, the Hela cells were xed with xative solution for 10 min and washed with PBS twice for 3 min and stained with Hoechst 33258 reagent (Beyotime, China) for 5 min in the dark. The cells were then washed twice with PBS for 3 min and then visualized using the IX73 microscope (Olympus, Japan).
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