Cells were transfected with scrambled siRNA or TIGAR siRNA and reseeded into 6-well plates around 24 h after transfection, followed by treatment with Vehicle, 10 μM or 20 μM of olaparib for additional 48 h. Cells were then subject to Annexin V/PI staining following manufacturer’s instruction (#640906, Biolegend). Briefly, cells were washed twice with PBS and stained with Annexin V and propidium iodide solution for 15 min at room temperature in the dark before analysis by flow cytometry.
Annexin 5 pi staining
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Annexin V/PI staining is a laboratory technique used for the detection and quantification of apoptosis, a type of programmed cell death. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is exposed on the outer cell membrane during the early stages of apoptosis. Propidium iodide (PI) is a fluorescent dye that binds to DNA and is used to identify cells with compromised cell membranes, which can occur in late apoptosis or necrosis. The combined use of Annexin V and PI allows for the differentiation of viable, early apoptotic, late apoptotic, and necrotic cells.
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6 protocols using annexin 5 pi staining
1
Olaparib-Induced Apoptosis in TIGAR-Knockdown Cells
2
Apoptosis Assay for Trichomonas Treated with TA and α-pinene
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The treated Trichomonas cells with TA (125–2000 μg/ml) and α-pinene (3.9–62.5 μg/ml) were assayed for apoptosis using annexin V/PI staining, according to the manufacturer’s kit (BioLegend, USA) instructions. Briefly, at the end of the 24 hours’ incubation (15 (link)), the cells were harvested by centrifuged at 2000 rpm for 5 min. The pellet was washed twice with phosphate buffer saline (PBS) and then resuspended in binding buffer at a concentration of 1.0 × 106 cells/ml. The cell suspension (100 μl) was transferred to a 5 ml test tube and 5 μl FITC annexin V and 10 μl PI were added, then the cells were gently vortexed and incubated at room temperature in the dark. After 15 min, 400 μl of annexin V binding buffer was added to the test tube and finally, analyzed by FACScalibur flow cytometer.
3
Evaluating SMIP34 Impacts on Cell Viability
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The effects of SMIP34 treatment on cell viability was assessed by using the MTT cell viability assay in 96-well plates as described [14 (link)]. Colony formation assays were done as described [14 (link)]. Briefly, BT-549 and MDA-MB-231 model cell lines (500 cells/well) were seeded in 6-well plates and allowed to grow for 5 days in control or SMIP34 treated medium and then medium was changed to normal medium and allowed to grow for 5–7 more days. Cells were then fixed in ice-cold methanol and stained with 0.5% crystal violet solution. The colony area percentage was calculated using NIH ImageJ software. Apoptosis was measured using Annexin V-PI staining (BioLegend, San Diego, CA) according to manufacturer’s protocol.
4
Combination Therapy Impacts TNBC Cell Viability
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The effects of vehicle (DMSO), EC359, and HDACi alone or in combination on cell viability were measured using the MTT cell viability assay16 (link). HDACi and EC359 concentrations used in the assays were based on our earlier studies16 (link), dose–response curves using TNBC models, and published studies15 (link). The combination index (CI) of EC359 + HDACi therapy was determined using Chou-Talalay method37 (link). Apoptosis was measured using Annexin V-PI staining (BioLegend, San Diego, CA) and Caspase-Glo® 3/7 assays (Promega, Madison, WI) according to manufacturer’s protocol. For colony formation assays, TNBC model cells (500 cells/well) were seeded in triplicate in 6-well plates, treated with indicated drugs for 5 days, and allowed to grow for 14 days. The cells were fixed in ice-cold methanol and stained with 0.5% crystal violet solution. Colonies that contain ≥50 cells were counted and used in the analysis. The effect of combination therapy on cell invasion was studied using the Corning® BioCoat™ Growth Factor Reduced Matrigel® Invasion Chamber assay (Corning, Corning, NY). MDA-MB-231 and BT-549 cells were treated with vehicle or EC359 or HDACi or combination for 22 h and invaded cells were determined and quantitated using the manufacturer,s protocol.
5
Evaluating Apoptosis in NP Cells
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The NP cells were cultured for 24 hours in complete medium and then treated with H2O2 (20 μM), with or without EET, in serum-free media for 4 hours. Microphotographs were taken under an Olympus BX51 microscope equipped with an Olympus DP70 digital camera (Olympus, Tokyo, Japan). Apoptosis of the NP cells was measured by annexin V/PI staining (Biolegend, San Diego, CA, USA) as previously described [36 (link)].
6
Splenic B Cell Isolation and Manipulation
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