The translocation of membrane phosphatidylserine from the inner side of the plasma membrane to the surface (Annexin V) and the membrane integrity (propidium iodide) was evaluated by the means of Apoptosis Detection Kit (Immunostep, Salamanca, Spain) according to the manufacturer’s instructions. MCF-7 cells were seeded in 6-well plates (1 × 106 cells/well) and incubated overnight. Cells were then treated with sphaerodactylomelol (IC50) for 24 h and stained with probes before analysis by flow cytometry. Untreated cells with DMSO were used as control. The antibiotic staurosporine (1 µg/mL) (Sigma, Rehovot, Israel) was used as positive control. Ten thousand events were attained with AMNIS imaging flow cytometer using the AMNIS INSPIRE™ software (Amnis Corporation v6.0, Luminex Corp, Austin, TX, USA). Data analysis was performed with the AMNIS IDEAS™ software. The results were expressed as percentage of events defined as viable, apoptosis, late apoptosis, and necrosis.
Apoptosis detection kit
Manufactured by Immunostep
Sourced in Spain
The Apoptosis Detection Kit is a laboratory assay designed to detect and measure apoptosis, a form of programmed cell death, in biological samples. The kit utilizes fluorescent probes to label and identify apoptotic cells, providing a quantitative assessment of the extent of apoptosis in the sample.
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7 protocols using apoptosis detection kit
1
Apoptosis and Membrane Integrity Analysis in MCF-7 Cells
2
Annexin V-FITC/PI Apoptosis Assay
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The PCD of the Raw 264.7 cells and the patient isolated monocytes were monitored by flow cytometry using double staining with Annexin V-fluorescein isothiocyanate and propidium iodide (PI; Apoptosis Detection kit; Immunostep, Salamanca, Spain), according to the manufacturer's instructions. The fluorescence was measured with a Cyan ADP cytometer and Summit version 4.3 software (Beckman Coulter, Fort Collins, CO, USA), and was subsequently analyzed with FlowJo 7.6 software (TreeStar Inc., Ashland, OR, USA). This technique was used to assess the effect of the treatments on cell viability. Debris were excluded from the plot based on the scatter (FSC vs. SSC) and the apoptotic (Annexin V positive, A+; PI negative, PI− and positive, PI+) and the necrotic (A− and PI+) cells were characterized based on the fluorescence emitted.
3
Apoptosis Analysis of MCF-7 Cells
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MCF-7 cells (1.0×106 cells/mL) were seeded in 6-well plates and treated with the compounds at the IC50 concentration and staurosporine (1 µg/mL) (Sigma, Rehovot, Israel) for 24 h, and the effects were analyzed by flow cytometry using the Apoptosis Detection Kit (Immunostep, Salamanca, Spain). Ten thousand events were recorded with the AMNIS imaging flow cytometer using the AMNIS INSPIRE™ software. The data were analyzed using the AMNIS IDEAS™ software (Amnis Corporation v6.0, Luminex Corp, Austin, TX, USA).
4
Annexin V-PI Apoptosis Assay
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The programmed cell death of Raw 264.7 cells was monitored by flow cytometry using double staining with Annexin V-FITC and Propidium Iodide (Apoptosis Detection Kit, Immunostep, Salamanca, Spain), according to the manufacturer’s instructions.
Fluorescence was acquired with Cyan ADP cytometer and Summit software (Beckman Coulter, Fort Collins, CO, USA) and then analysed with FlowJo software (version7.6, TreeStar Inc., Ashland, OR, USA). This technique was used to assess the effect of treatments on cell viability. Debris were excluded from the plot based on the scatter (FSC vs. SSC), and apoptotic (Annexin V positive, A+; Propidium Iodide negative PI− and positive PI+) and necrotic (Annexin V negative, A−; Propidium Iodide positive PI+) cells were characterized based on the fluorescence emitted.
Fluorescence was acquired with Cyan ADP cytometer and Summit software (Beckman Coulter, Fort Collins, CO, USA) and then analysed with FlowJo software (version7.6, TreeStar Inc., Ashland, OR, USA). This technique was used to assess the effect of treatments on cell viability. Debris were excluded from the plot based on the scatter (FSC vs. SSC), and apoptotic (Annexin V positive, A+; Propidium Iodide negative PI− and positive PI+) and necrotic (Annexin V negative, A−; Propidium Iodide positive PI+) cells were characterized based on the fluorescence emitted.
5
Apoptosis Detection by Flow Cytometry
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Apoptosis was detected by Annexin V-FITC/Propidium Iodide staining using an apoptosis detection kit (Immunostep) according to the manufacturer's protocol. 15 Briefly, cells were harvested by trypsin, washed twice with temperate PBS, and suspended in 1X Annexin Buffer at a concentration of 1 × 10 6 cells/ml. One hundred microliter of cell suspension were added with 5 µl of Annexin V-FITC and 5 µl of Propidium Iodide (PI). Cells were incubated at room temperature for 15 min in the dark. Subsequently, 400 µl of 1X Annexin Buffer were added to each sample. Samples were analyzed by flow cytometry.
6
Measuring Cell Death by Flow Cytometry
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The cells were maintained in culture with or without treatment for 48–72 hours (Table S1 ). Then, both suspended cells and adherent cells were collected, and the ‘Apoptosis Detection’ kit (Immunostep) was used to measure cell death following the manufacturer’s instructions. Briefly, cells were stained with Annexin V for 15 min at room temperature and in darkness. Subsequently, they were washed with Binding Buffer solution and centrifuged to remove the supernatant. Finally, they were incubated with propidium iodide for 5 min at room temperature. A FACSCanto II flow cytometer (BD Biosciences) was employed to detect the staining, and the results were analyzed using Diva software. Cells stained only with Annexin V were considered in early apoptosis, cells stained only with propidium iodide were considered in necrosis, and cells stained with both markers were considered in late apoptosis. All cells stained with propidium iodide were considered dead (Supp. Figure 11 ).
7
Quantifying Programmed Cell Death
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Programmed cell death was evaluated on both microglial and neuronal cells. PCD were evaluated by flow cytometry using Annexin V and Propidium Iodide staining (Apoptosis Detection Kit, Immunostep, Salamanca, Spain), as described elsewhere [6] . PCD is expressed as a percentage of Annexin V positive cells (A+). In standard contact coculture, staining with CellTrace Far Red allowed to evaluate PCD simultaneously on BV-2 cells and on unstained SH-SY5Y.
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