Apoptosis assay cell type - Human endometrial stromal cells

A gross majority of classical apoptotic attributes can be quantitatively examined by flow cytometry, the preferred platform for rapid assessment of multiple cellular attributes at a single-cell level. However, sample preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

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Cell Death Detection ELISA

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	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.

Protocol tips
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.

Protocol tips

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.

Manufacturer protocol Publication protocol 1 Relevant paper

FragEL™ DNA Fragmentation Detection Kit, Colorimetric - TdT Enzyme

Millipore

Upstream tips	Protocol tips	Downstream tips
	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.

Protocol tips
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.

Protocol tips

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.

Manufacturer protocol Publication protocol 1 Relevant paper

In situ apoptosis detection

Takara Bio Inc

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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.

Protocol tips
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.

Manufacturer protocol Publication protocol 1 Relevant paper

QIA33 | FragEL™ DNA Fragmentation Detection Kit, Colorimetric - TdT Enzyme

Millipore

Upstream tips	Protocol tips	Downstream 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.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

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