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Found 4 matching solutions for this experiment
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Cell apoptosis was detected using a photometric enzyme immunoassay (Cell Death Detection ELISA kit, cat. no. 11544675001; Sigma-Aldrich Merck KGaA), as previously described (30). This is based on the quantitative sandwich immunoassay employing antibodies against histone and DNA. MDA-MB-231 cells and MCF-7 cells (1×105 cells/well) were seeded into a 6-well plate in the presence or absence of different concentrations of BBM (10, 20 and 40 µM) and cultured for 24 h at 37°C. The cells were washed with PBS and then lysed in RIPA lysis buffer (containing 1 mM PMSF). The supernatant was collected via centrifugation at 12,000 × g for 15 min at 4°C. and used for testing. The mono- and oligonucleosomal fragmented DNA was measured according to the instructions of the manufacturer. |
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| Cell apoptosis was detected using a photometric enzyme immunoassay (Cell Death Detection ELISA kit, cat. no. 11544675001; Sigma-Aldrich Merck KGaA), as previously described (30). This is based on the quantitative sandwich immunoassay employing antibodies against histone and DNA. MDA-MB-231 cells and MCF-7 cells (1×105 cells/well) were seeded into a 6-well plate in the presence or absence of different concentrations of BBM (10, 20 and 40 µM) and cultured for 24 h at 37°C. The cells were washed with PBS and then lysed in RIPA lysis buffer (containing 1 mM PMSF). The supernatant was collected via centrifugation at 12,000 × g for 15 min at 4°C. and used for testing. The mono- and oligonucleosomal fragmented DNA was measured according to the instructions of the manufacturer. |
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For the in vivo apoptosis assay, eutopic endometrial tissue sections were fixed in 4% paraformaldehyde for 24 h at 4°C, washed in phosphate-buffered saline (PBS), and embedded in paraffin blocks. The tissue sections were incubated with proteinase K (20 μg/mL, without DNAase), washed three times with HBSS, and subsequently labeled using the TdT-FragEL™ DNA fragmentation detection kit (Calbiochem), according to the manufacturer’s instructions. |
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| For the in vivo apoptosis assay, eutopic endometrial tissue sections were fixed in 4% paraformaldehyde for 24 h at 4°C, washed in phosphate-buffered saline (PBS), and embedded in paraffin blocks. The tissue sections were incubated with proteinase K (20 μg/mL, without DNAase), washed three times with HBSS, and subsequently labeled using the TdT-FragEL™ DNA fragmentation detection kit (Calbiochem), according to the manufacturer’s instructions. |
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To assess any potential toxicity of the miR-542-3p mimic transfection, apoptosis and necrosis were quantified by direct determination of nucleosomal DNA fragmentation using the Apoptotic/Necrotic/Healthy Cells Detection Kit (Takara bio Inc., Japan) as per the manufacture’s instructions. |
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| To assess any potential toxicity of the miR-542-3p mimic transfection, apoptosis and necrosis were quantified by direct determination of nucleosomal DNA fragmentation using the Apoptotic/Necrotic/Healthy Cells Detection Kit (Takara bio Inc., Japan) as per the manufacture’s instructions. |
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- Tissues were fixed in 4% paraformaldehyde for 24 h at 4°C,
- treated with proteinase K
and Incubated at room temperature for 20 min. |
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| Protocol tips |
- Tissues were fixed in 4% paraformaldehyde for 24 h at 4°C,
- treated with proteinase K
and Incubated at room temperature for 20 min. |
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