Cells were plated in complete media in 24-well plates prior to starvation. Twenty-four hours after seeding, cells were washed with PBS and incubated in the indicated glutamine- or serum-starvation media with 10% dialyzed FBS. For rescue experiments, the cells were incubated in the media supplemented with either 0% BSA or 4% BSA. Negative control cells for macropinocytosiswere treated with EIPA. The number of viable cells was assessed using automated cell proliferation detector using IncuCyteTM (Essen Instruments, Ann Arbor, MI, USA). Proliferation was measured through quantitative kinetic processing metrics derived from time-lapse image acquisition and presented as percentage of culture confluence over time. The number of cells wasalso counted either using a Coulter Counter (Beckman Coulter, Brea, CA, USA) and/or using trypan blue exclusion and an automated cell counter (Nano-Entek, Seoul, Korea). Cell viability was determined by Annexin V and propidium iodide (PI) staining following standard protocols at indicated periods of time (BD Biosciences). Cells negative for both Annexin V and PI staining were considered live cells.
Annexin 5 and propidium iodide
Manufactured by BD
Sourced in United States
Annexin V and propidium iodide are fluorescent dyes used in cellular and molecular biology applications. Annexin V binds to the phospholipid phosphatidylserine, which is exposed on the surface of apoptotic cells. Propidium iodide is a DNA-binding dye that can only penetrate cells with compromised membranes, such as necrotic or late-stage apoptotic cells. Together, these dyes can be used to discriminate between viable, apoptotic, and necrotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Automated cell counter, by NanoEnTek (2 mentions)
Coulter counter, by Beckman Coulter (2 mentions)
Facsdiva software v6, by BD (1 mentions)
Canto 2, by BD (1 mentions)
Sulforhodamine b srb, by Merck Group (1 mentions)
Flow cytometry, by BD (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Cellquest software, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Annexin 5, by BD (1 mentions)
Facsaria fusion, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Dexamethasone (dex), by BD (1 mentions)
Celltracker green cmfda, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Media matrigel, by BD (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
20 protocols using annexin 5 and propidium iodide
1
Quantifying Cell Viability and Proliferation
2
Immunosuppression Effects on NK Cells
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The effect of immunosuppressive compounds on apoptosis and necrosis of human NK cells was assessed by means of Annexin V and propidium iodide (PI) staining (BD Biosciences). NK cells were incubated with or without the immunosuppressive agent for 24 h and subsequently labeled according to manufacturer´s instructions. The percentages of annexin+PI- (apoptotic) and annexin+PI+ (necrotic) NK cells, respectively, were assessed by flow cytometry (Canto II, Beckton Dickinson; BD FACSDiva Software v6.1.3, BD Biosciences). Each experiment has been performed at least three times, and all experiments have been performed independently from each other using NK cells from a different donor.
3
Cell Viability Assay Protocol
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Cells were plated in complete media on either 96 well or 24 well plates prior to starvation. Twenty-four hours after seeding, cells were washed with PBS and incubated in the indicated glutamine-or EAA starvation media with 10% dialyzed FBS. For rescue experiments, the cells were incubated in the media supplemented with 2% BSA. Negative control cells for macropinocytosis were treated with EIPA. The number of viable cells was assessed using the MTT assay and relative cell number was determined by sulforhodamine B (SRB; Sigma-Aldrich) staining following standard protocols at indicated periods of time to examine the cytotoxic effect of the drug in vitro. The number of cells was counted either using a Coulter Counter (Beckman Coulter) and/or using trypan blue exclusion and an automated cell counter (Nano-Entek, Korea). Cell viability was determined by Annexin V and propidium iodide (PI) staining following standard protocols at indicated periods of time (BD Biosciences). Cells negative for both Annexin V and PI staining are considered live cells.
4
TRIM72 Overexpression Impacts Cell Viability, Cycle, and Apoptosis
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BT549 and MDA-MB-231 cells were transfected with TRIM72-oe or NC plasmids in 6-well plates. Approximately 24 h after transfection, the cells (2 × 103 cells) were seeded in a 96-well plate, and cell viability was measured using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, Wisconsin, USA) every 24 h, thrice. For cell cycle studies, cells transfected with TRIM72 overexpression plasmid or NC for 24 h were harvested using 0.25% trypsin. Then, 1 × 106 cells were fixed with 70% ethanol for 30 min at 4 °C and stained using a Cell Cycle Kit (Forevergen Biosciences Co., Ltd., Guangzhou, China) in the dark for 30 min. Subsequently, the cells were washed with PBS and subjected to flow cytometry (BD Biosciences, San Jose, California, USA). For cell apoptosis analysis, cells were harvested 24 h after transfection, washed with PBS twice, resuspended in binding buffer, and double-stained using Annexin V and propidium iodide (BD Biosciences). Apoptosis analysis was performed by flow cytometry.
5
Annexin V and PI staining for apoptosis
6
Apoptosis in RL95-2 and HEC-1A Cells
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The RL95-2 and HEC-1A cells were seeded in 6-well flat-bottom microplates at the density of 3×105 cells/well, and treated with NOMAC (0, 10, 30, and 100 μM) or metformin (1 mM), NOMAC (30 μM), or NOMAC (30 μM) plus metformin (1 mM) for 48 h. Then cells were digested by 0.25% trypsin with EDTA (Ethylene Diamine Tetraacetic Acid) and centrifuged at 800 rpm for 5 min. After resuspension with 100 μL binding buffer and labeling by Annexin V and Propidium iodide (BD Biosciences, San Jose, CA, USA), a BD LSRFortessa flow cytometer (BD Biosciences) was used to detect cell apoptosis and the percentage of apoptotic cells was analyzed using Flowjo software (Tree Star, Ashland, OR, USA).
7
Neutrophil Survival in CARD9 Deficiency
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Neutrophils from healthy donors and the CARD9-deficient patient were harvested as described above. To determine whether CARD9 deficiency impairs survival of neutrophils, cells were cultured for 3 or 6 hours at 37°C in a 5% CO2 incubator in RPMI 1640 without FBS (serum starvation) or in RPMI 1640 with 10% FBS with tumor necrosis factor-α (50 ng/mL) or live Candida albicans SC5314 (1 x 105 cells/mL) or in infected CSF from the CARD9-deficient patient to assess the extent of cell apoptosis and death. Cells were stained with Annexin V and propidium iodide (BD Biosciences) per the manufacturer’s instructions, and flow cytometry was performed on a 5-laser LSRFortessa.
8
Evaluating Apoptosis in Macrophages and Endothelial Cells
9
Apoptosis Induction in Leukemia Cells
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K562/A02 and HL-60/ADM were plated in six-well plates (6×105 cells/well). After 24 h transfection, K562/A02 and HL-60/ADM cells were treated with Ara-C at a final concentration of 5 μM. After 48 h of treatment with Ara-C, the cells were stained with Annexin V and propidium iodide (BD Biosciences, San Jose, CA, USA) and then analyzed through flow cytometry (BD FACSAria™ Fusion).
10
Measuring Spontaneous Apoptosis in TNBC
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The effect of TLN1 silencing on spontaneous apoptosis in TNBC cells was measured by flow cytometry using an Annexin-V and propidium iodide (PI) kit (559763, BD, San Diego, CA), following the protocol provided. Briefly, the different groups of cells (2 × 105/tube) were stained in duplicate with 5 µl of Annexin V-FITC and PI in the dark for 15 min. The cells were then analysed by flow cytometry (BD FACSverse, Piscataway, NJ ).
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