We performed in vitro experiments to demonstrate drug elimination of suicide gene-modified cells through activation of the suicide gene of interest. The following drugs were applied at the indicated concentrations unless otherwise stated: non-therapeutic chemical inducer of dimerization, BB homodimerizer, [100nM] (AP-20187, Clontech; Mountain View, CA), sirolimus 25 ng/mL, or rituxan 100 ug/mL in the presence of rabbit serum complement (Innovative research, Novi, MI). Cells were incubated overnight with chemical inducer, except in experiments using rituxan, where the incubation time was 4 hours. After the appropriate treatment, cells were washed and stained for viability and apoptosis using the Annexin V/7AAD kits (BD Biosciences). We added 123e counting beads (Invitrogen) and acquired a constant number of beads for each experimental condition using the FACS Canto II. The degree of cellular’s elimination was estimated using the following formula: [100% - (%Viability treated/% Viability non-treated cells)]. To confirm that killing was due to apoptosis, we performed some experiments after pretreatment with 20 uM of the pan-caspase inhibitor Z-VAD-FMK for 1 hour (BD Pharmingen).
Annexin v 7 aad kit
Manufactured by BD
Sourced in United States
The Annexin V/7-AAD kit is a fluorescent reagent system used for the detection of apoptosis in cell samples. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is exposed on the outer membrane of apoptotic cells. 7-AAD is a nucleic acid dye that can only penetrate cells with compromised cell membranes, such as those undergoing late-stage apoptosis or necrosis. The kit allows for the simultaneous detection of early and late apoptotic cells in a sample.
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Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Compusyn software, by ComboSyn (2 mentions)
Z vad fmk, by BD (1 mentions)
Ap20187, by Takara Bio (1 mentions)
Facscanto 2, by Thermo Fisher Scientific (1 mentions)
Peroxidase affinipure donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
β actin antibody a5441, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (1 mentions)
Peroxidase affinipure donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Prestoblue, by Thermo Fisher Scientific (1 mentions)
Facscanto system, by BD (1 mentions)
Annexin 5 and 7 aad, by BD (1 mentions)
Annexin 5 fluos staining kit, by Roche (1 mentions)
Accuri c6 plus, by BD (1 mentions)
Cd117 c kit apc, by Thermo Fisher Scientific (1 mentions)
Cd90 fitc, by Dianova (1 mentions)
Facsaria 2, by BD (1 mentions)
Cfse cell division tracker kit, by BioLegend (1 mentions)
14 protocols using annexin v 7 aad kit
1
In Vitro Suicide Gene Elimination
2
Cell Cycle and Apoptosis Analysis
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The cell cycle and apoptosis was analysed by flow cytometry (FACSCanto II, BD Biosciences) using PI staining or Annexin V/7-AAD kits (BD Biosciences) according to the standard protocol.
3
Cell Cycle and Apoptosis Analysis
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The HuH7 and HCCLM3 cell were seeded in six well plates and transfected with p-miR-1258 and p-miR-control plasmids as mentioned above. 48 hour after transfection, the cells were collected for cell cycle and apoptosis analysis. The cell cycle and apoptosis was analysed by flow cytometry (FACSCanto II, BD Biosciences) using PI staining (for cell cycle) or Annexin V/7-AAD kits (for apoptosis) (BD Biosciences) according to the standard protocol.
4
Targeting TIE-1 and PI3K Signaling
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Peroxidase AffiniPure Donkey Anti-Rabbit IgG (#711-035-152) and Peroxidase AffiniPure Donkey Anti-Mouse IgG (#715-035-150) were from Jackson ImmunoResearch (West Grove, PA, USA). Antibodies specific to TIE-1 (ab111547) and PTEN (ab32199) were obtained from Abcam (Cambridge, MA, USA). Other antibodies for phospho-Akt (S473, #9271) and PI3K p110α (#4249) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies specific to V5 (#R960-25) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The β-actin antibody (A5441) was obtained from Sigma (St Louis, MO, USA). RNAiMax; Lipofectamine 2000; and siRNAs for control (4390846), TIE-1 (s14140 and s14141), and PI3K (s10521) were from Life Technologies (Carlsbad, CA, USA). The CellTiter-Glo luminescent cell-viability-assay kit was obtained from Promega (Fitchburg, WI, USA). The annexin V/7-AAD kit was from BD Biosciences (San Jose, CA, USA).
5
Evaluating Compound Cytotoxicity and Apoptosis
6
Cell Viability and Apoptosis Assay
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Cells were seeded in a 96-well plate and treated with the drug or DMSO for 48 h. PrestoBlue (ThermoScientific, USA) was used to measure cell viability following the manufacturer’s protocol. For apoptosis assay, cells were seeded in a 24-well plate and treated with the drug or DMSO for different time points. Apoptotic cells were quantified using the annexin-V/7-AAD kit (BD Biosciences, USA) following the manufacturer’s protocol.
7
Annexin V/7AAD Apoptosis Assay
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Apoptosis was examined by human Annexin V/7AAD kit (BD Biosciences, USA) according to the manufacturer's instructions. Briefly, cells (2.5×105) seeded in 6-well plates were treated with t-BHP with or without Z-VAD-FMK (1 µM) pretreatment. Then cells were harvested by incubation with Annexin V/FITC solution for 15 min at room temperature. The samples were immediately analyzed by flow cytometry using a FACSCantoTM system (BD Biosciences, USA). At least 1×104 cells were analyzed for each sample.
8
Apoptosis induction by Cucurbitacin D in cervical cancer
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The apoptosis inducing effect of Cucurbitacin D on cervical cancer cells was analyzed by Annexin V-FLUOS staining kit (Roche Diagnostic Corp., Indianapolis, IN). In this experiment, 60% confluent CaSki and SiHa cells were treated with Cucurbitacin D for 24 hrs. Cells were washed with PBS (1X) and kept in DAPI and Annexin-V solution for 20 min. Images were captured under fluorescence microscope. Apoptosis was further determined by Annexin V-7AAD kit (BD Biosciences). In this experiment, SiHa cells (1 × 106) were plated in a 100 mm dish and allowed to adhere overnight. The next day, cells were treated with 0.5 and 1.0 μM Cucurbitacin D or equivalent amounts of controls for 24 hrs. Both adherent and floating cells were collected and stained with Annexin V and 7-AAD (BD Biosciences) at 5 μL of each/100 μL of cell suspension. Cells were incubated for 20 min in the dark at room temperature and analyzed with an Accuri C6 Flow Cytometer in FL2 and FL3 channels.
9
Annexin V and 7-AAD Apoptosis Assay
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The cultured cells were stained with phycoerythrin labelled Annexin V/7-AAD (7-aminoactinomycine-D) according to the manufacturer's instructions (Annexin V/7-AAD kit, BD Biosciences, US). The cell pellet was washed and resuspended in 100 µL binding buffer at a concentration of 10 5 -10 6 cells/mL; 5 µL of Annexin V together with 5 µL 7-AAD was added to 100 µL cell suspension. MCF-7 cells were incubated for 15 minutes in the dark [30] (link). The samples were analyzed by ow cytometry (BD Accuri C6 Plus, US), and data were recorded as the percentage of the cells in four quadrants as follows; Top-right: Late apoptosis, Top-left: Necrosis, Bottom-right: early apoptosis, Bottom-left: Living cells.
10
Annexin V/7-AAD Apoptosis Assay
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Apoptotic cells was determined by Annexin V/7 AAD kit (BD Biosciences) according to the manufacturer’s protocol.
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