To perform our apoptosis analyses, we trypsinated the Neuro2A cells and stopped the enzyme with media-containing serum. In addition, we kept all the washes to not loose dead cells. After centrifugation, the supernatant was discarded, and cell pellets were resuspended in PBS and transferred into micro centrifuge tubes. In order to have a positive control for early and late apoptosis, the untransfected cell condition was used. Half of the cells were killed intentionally for 15 min at 75°C. These cells were then mixed with the rest of untransfected cells in equal parts and spun down. Once the positive apoptosis control was made, all the cell suspensions were spun down and resuspended into a 100 μl Annexin V-binding buffer (BD Biosciences). Annexin V (V450, 560506 BDBiosciences) and 7-aminoactinomycin D (7AAD) (559925 BD Biosciences) were added to analyze for early and late apoptosis, excluding the single-stain controls. The single-stain controls were the positive apoptosis control either incubated with Annexin V or 7AAD. Incubation was performed for 15 min at room temperature in the dark. After the incubation, an Annexin V-binding buffer was added, and cells were passed through tubes with a 35-mm strainer (VWR) and analyzed with the SH800 Cell Sorter (Sony).
Annexin 5 v450
Manufactured by BD
Sourced in United States
Annexin V-V450 is a fluorescent conjugate of the Annexin V protein, which binds to phosphatidylserine. It is primarily used in flow cytometry applications to detect and quantify apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 binding buffer, by BD (5 mentions)
7 aminoactinomycin d, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Lsrfortessa cell analyzer, by BD (2 mentions)
Rnase a, by Merck Group (2 mentions)
7 aminoactinomycin d 7 aad, by Thermo Fisher Scientific (2 mentions)
Lsr 2, by BD (2 mentions)
Sh800 cell sorter, by Sony (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Methanol, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Etoposide, by Merck Group (1 mentions)
Z vad fkm, by Merck Group (1 mentions)
7 aad, by BD (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Arpe 19, by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Arpe 19, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Sk n as, by Thermo Fisher Scientific (1 mentions)
16 protocols using annexin 5 v450
1
Annexin V-based Apoptosis Assay
2
Analysis of Apoptosis in Transfected HeLa Cells
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Transfected HeLa cells were harvested, counted, washed twice in PBS and 1 × 106 cells were stained for 30 min with 1 μl 780eFluor of Fixable Viability Dye (eBioscience) in 1 ml PBS. For Z-VAD-FKM experiments, culture media was removed 6 h after transfection and replaced with 20 μM Z-VAD-FKM (Sigma) containing complete media. After PBS washing, cells were stained with 10 μl Annexin V-V450 (BD Biosciences, 560506) in 200 μl 1× binding buffer for 15 min. After washing, cells were fixed in 2% ice-cold paraformaldehyde (Electron Microscopy Sciences) for 15 min and permeabilized in 90% ice-cold methanol (Sigma-Aldrich) for 30 min. V5 staining was then performed using mouse anti-V5 Alexa-Fluor 647 conjugated antibody (clone SV5-Pk1, Biorad) and stained samples were acquired on a MACSQuant Analyzer (Miltenyi Biotec). Data were analyzed with FlowJo software (Tree Star Inc). Treatment with 100 μM etoposide (Sigma) in dimethylsulfoxide for 12 h was used as positive control.
3
Apoptosis Analysis of M-CSF Macrophages
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Proportions of apoptotic cells were analysed from macrophages that were differentiated with M‐CSF and in the absence or presence of 50 or 500 µg/ml hLF. Macrophages were stained with annexin V‐V450 and 7‐aminoactinomycin D (7‐AAD) viability staining solution (both from BD Biosciences) and analysed subsequently by flow cytometry.
4
Cell Cycle and Apoptosis Analysis by Flow Cytometry
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For cell cycle analysis, a total of 5×105 cells were collected and fixed with 70% ethanol overnight. Cells were subsequently stained in dark at room temperature for 15 min with PBS buffer containing 25 µg/ml 7-amino-actinomycin D (BD Biosciences, San Jose, CA) and 10 µg/ml RNase A (Sigma Aldrich) and 0.1% Triton X-100 prior to flow cytometric analysis. For detection of apoptotic cells, cells were washed with cold PBS and re-suspended in 1x Annexin V binding buffer (BD Biosciences) at a concentration of 1×106 cells/ml. Annexin V-V450 (BD Biosciences) and 7-aad (BD Biosciences) were used according to the manufacturer’s instruction. For GFP-expressing cell population analysis, raw cells and LZRS virus-infected cells were co-cultured for 24 h and 72 h prior to flow cytometric analysis. BD LSRFortessa cell analyzer (BD Biosciences) and BD FACSDiva™ software (BD Biosciences) were used for flow cytometry and analysis of cell distribution, respectively.
5
Culturing Retinoblastoma and Neuroblastoma Cells
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Human retinoblastoma cell line Y79RB and retinal pigmental epithelial cell line ARPE-19 were purchased from American Type Culture Collection (ATCC, HTB-18 and CRL-2302, respectively, Manassaas, VA, USA). Human neuroblastoma cell line SK-NAS was a gift from Dr. Naravat Poungvarin, Faculty of Medicine Siriraj Hospital, Mahidol University. The Y79RB cells were cultured in suspension in Roswell Park Memorial Institute (RPMI) 1640 Medium (GibcoⓇ Life Technologies, Thermo Fisher Scientific, CA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA, USA), 100 IU/ml penicillin, 100 μg/ml streptomycin (Mediatech; Corning, NY, USA). For ARPE-19, SK-NAS and primary NB cells, cells were maintained in Dulbecco's Modified Eagle Medium /Nutrient Mixture F-12 (DMEM/F12) (GibcoⓇ Life Technologies, Thermo Fisher Scientific, CA, USA). All cells were maintained in a 37 °C incubator with 5%CO2. Recombinant human cytokines, including interleukin (IL)−2 (250 µg/ml), IL-7 (250 µg/ml), and IL-15 (250 µg/ml), were purchased from PeproTech (PeproTech, Cranbury, CT, USA). AnnexinV-V450 and propidium iodide (PI) were purchased from BD Pharmingen (BD Biosciences, San Jose, CA, USA).
6
Immunophenotyping of Hematopoietic Cells
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Annexin V-V450, anti-CD138-APC, anti-CD41a-PE, anti-P-gp-FITC, anti-CD34-PE-Cy7, matched isotype controls, BDTM CompBeads anti-mouse-Ig k, SpheroTM Rainbow calibration particles and TruCountTM tubes were from BD Biosciences (Sydney, NSW, Australia). Latex beads of 0.3 and 1.1 µm diameter were from Sigma-Aldrich (Sydney, NSW, Australia). Details of other reagents used are described in Supplementary Table 1 .
7
Profiling Cancer Stem Cell Markers
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Dissociated cancer cells were filtered through a 4 µm strainer and suspended in PBS supplemented with 2% FBS and 2 mM EDTA (FACS buffer) as previously described [73 (link)]. One µL of mouse IgG (1 mg/mL) was added and incubated at 4 °C for 10 min. The cells were then re-suspended in 1× binding buffer and anti-CD44 (APC) in combination with anti-CD24 (PE) antibodies (BD, Mississauga, ON, Canada) according to the manufacturer’s instructions for 30 min. The cells were washed twice with FACS buffer and 7-aminoactinomycin D (7-AAD, eBioscience, San Diego, CA, USA) and Annexin-V/V450 (BD) was added and incubated for 15 min at room temperature to assess dead and apoptotic cells. Flow cytometry was performed on the BD LSRFortessa. Data were analyzed with FlowJo software (Ashland, OR, USA).
8
Annexin V and Caspase-3 Apoptosis Assay
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Apoptosis assays were performed according to the manufacturer’s protocol using the annexin V apoptosis detection kit (eBioscience) and annexin V-V450 (BD Horizon) and propidium iodide (eBioscience). For intracellular staining of cleaved caspase 3, cells were fixed with 1.5% formaldehyde for 10 minutes at room temperature, permeabilized with 100% ice-cold methanol for 20 minutes on ice, washed twice with staining buffer and incubated with antibodies.
9
Quantifying Myeloma Cell Surface Markers
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Annexin V-V450 (BD Horizon, cat. no.560506), anti–CD138-APC (clone MI15; cat. no. 347193), anti–CD41a-PE (clone HIP8; cat. no. 555467), TruCount tubes (cat. no. 340334), and matched isotype controls were purchased from BD Biosciences Australia/New Zealand. Latex beads of diameter 0.3 μm (cat. no. LB3) and 1.1 μm (cat. no. LB11) were from Sigma-Aldrich, Australia. Australian Microscopy and Microanalysis Research Facility at the Australian center provided all consumables for electron microscopy. Myeloma cell line OPM2 was kindly provided by Royal Prince Alfred Hospital Haematology, Sydney, Australia, and was tested for mycoplasma at University of Technology Sydney before use.
10
Annexin V and 7-AAD Apoptosis Analysis
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