Culture supernatants were collected, and after centrifuging at 300 g for 5 min and 2000 g for 20 min, EVs were bound to antibody-coated beads that had been blocked with 0.1% BSA (Sigma) in PBS for 30 min. According to our previous experiments, 20 µl or 6 µl of anti-CD63-coated beads (Thermo Fisher, 10606D) or anti-CD81-coated beads (Thermo Fisher, 10616D) were added to 200 µl supernatant, respectively. After overnight rotation at 4 °C, beads were magnetically separated, washed with Annexin binding buffer (BD Biosciences) three times and beads were labelled for 20 min. 10,000 beads were then measured on a FACSCalibur (BD) instrument. FITC-anti-CD81 (A15753, Molecular Probes), PE-anti-CD63 (SAB47000218, Sigma), and FITC Annexin V (SAB4700218, Sony) or APC Annexin V (Immunotools, 31490016) were used for detection (2 µl/sample). In all experiments, cells were counted with Burker chamber and results were normalized to cell number.
Annexin 5 apc
Manufactured by Immunotools
Sourced in Germany
Annexin V-APC is a fluorescent conjugate used to detect and quantify apoptotic cells. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is externalized during the early stages of apoptosis. The APC (allophycocyanin) fluorescent dye is conjugated to Annexin V, allowing for the detection and analysis of apoptotic cells by flow cytometry or fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (6 mentions)
Facsdiva 6, by BD (4 mentions)
Facscanto 2, by BD (3 mentions)
Annexin 5 binding buffer, by BD (3 mentions)
Facscanto 2 flow cytometer, by BD (3 mentions)
Flowlogic, by Miltenyi Biotec (3 mentions)
Cytoflex flow cytometer, by Beckman Coulter (3 mentions)
Facscalibur, by BD (2 mentions)
Kaluza 1.5a software, by Beckman Coulter (2 mentions)
Accutase solution, by Merck Group (2 mentions)
Enspire 2300 multimode plate reader, by PerkinElmer (2 mentions)
Annexin binding buffer, by BD (1 mentions)
Anti cd63 coated beads, by Thermo Fisher Scientific (1 mentions)
Fitc annexin 5, by Sony (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Bptes, by Merck Group (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions)
Facscanto, by BD (1 mentions)
Anti cd150 pe, by R&D Systems (1 mentions)
22 protocols using annexin 5 apc
1
EV Characterization via Flow Cytometry
2
Multicolor Flow Cytometry of BMDE and BMDM
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BMDE or BMDM were harvested and washed once in FACS buffer (PBS/2% FCS/1 mg/ml sodium azide), incubated with anti-CD16/CD32 blocking mAb (2.4G2, BioXcell) for 5 min at room temperature, and stained with diluted mAb mixtures. The following mAbs were used: PE- or Alexa647-coupled anti-mouse-Siglec-F (clone E50-2440, BD Biosciences, San Jose, CA) to determine BMDE purity and Alexa488-coupled anti-mouse-CD11b and APC/Cy7-coupled anti-mouse-F4/80 to determine BMDM purity. Apoptotic cells were stained with APC-Annexin V (Immunotools) in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2) according to manufacturer’s protocol. Propidium iodide (eBioscience, San Diego, CA) was added to identify dead cells. Samples were acquired on a FACS Canto II instrument (BD Immunocytometry Systems, San Jose, CA) and analyzed by FlowJo software (Tree Star, Ashland, OR).
3
Mouse and Human CD8+ T Cell Viability
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To measure the viability, 10 5 mouse or human memory CD8 + T cells were plated in a 96-well plate in RPMI medium. The cells were incubated at 37 C for up to 7 days. The cells were washed in Annexin V Binding Buffer (BD PharMingen) and stained with APC Annexin V (ImmunoTools) and PI (Sigma Aldrich). Samples were acquired on an AccuriÒ C6 Flow Cytometer and analyzed with FlowJo-Software (FlowJo 10.2). Viable cells were defined as Annexin V and PI double negative. Where indicated, cells were incubated in presence of BPTES (Sigma Aldrich) at 50 mM or in glutamine-free RPMI containing dialyzed FCS (Life Technologies).
4
Apoptosis of Mesothelioma Cells in Coculture
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M14K mesothelioma cells (2 × 106 cells) were labeled with 5 mM of carboxyfluorescein succinimidyl ester (CFSE, Sigma Aldrich, St. louis, MO, USA) in 1 mL of DMEM during 7 min at 37 °C in the presence of 5% CO2. THP-1 monocytes (1 × 105 cells in 24-well plate) were untreated (mock) or treated with PMA (16.2 µM), VPA (2.5 mM), or EPZ (10 µM) for 48 h. After washing, THP-1 monocytes were mixed with CFSE-labeled M14K cells (1 × 104 cells) and further cultured for 48 h. Primary monocytes (2 × 105 cells in a 24-well plate) and CFSE-labeled M14K cells (10 × 103 cells) were cocultured for 24 h in the presence of VPA (2 mM). Floating and adherent cells were collected, washed with PBS, and resuspended in 100 µL of annexin binding buffer (10 mM Hepes, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4, BD Biosciences, Franklin Lakes, NJ, USA). Cells were then labeled with annexin V-APC (5 µL, Immunotools, Friesoythe, Germany) during 15 min at room temperature. After adding 150 µL of annexin binding buffer, labeled cells were recorded by flow cytometry (BD FACSCanto, BD Biosciences, Franklin Lakes, NJ, USA). CFSE and annexin V double-positive events were considered as apoptotic M14K cells.
5
Cell Proliferation and Viability Assay
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Dye retention was analyzed by detecting CFSE fluorescence intensity in the live cell fraction. Viability was determined using Annexin V-APC staining. KG1a cells previously labeled with CFSE were collected from single cultures by pipetting. From cocultures, in addition to pipetting off the suspension cells, 200 μL of 1 mM ethylenediaminetetraacetic acid (EDTA) was also added to loosen the alginate. The cells were pelleted and resuspended in Annexin V buffer (10 mM HEPES/NaOH, pH 7.5, 140 mM NaCl, 2.5 mM CaCl2) containing 1.5 μL Annexin V-APC (ImmunoTools). The cells were incubated on ice in the dark for 15 min, followed by analysis by flow cytometry. The intensity of the CFSE signal was measured in the live cell fraction as a determinant of cell proliferation rate. For immunofluorescence labeling, primary AML blasts were harvested and resuspended in 1% bovine serum albumin (BSA) in PBS and blocked for 10 min after which the cells were collected and resuspended in 100 μL 1% BSA/PBS containing 2 μL of antibodies SLAMF1/CD150-PE (R&D Systems) or GRPC5C Alexa Fluor 405 (R&D Systems) and incubated on ice in the dark for 30 min. After washing of unbound antibodies with 400 μL of 1% BSA/PBS, the cells were resuspended in 200 μL of 1% BSA/PBS for measurement.
6
Sodium Alginate Hydrogel Preparation
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All materials were purchased from Sigma-Aldrich unless stated otherwise. 2% sodium alginate was prepared by dissolving sodium alginate in phosphate buffered saline (PBS) and sterilized by heating to 80°C in a water bath for 15 min.65 (link) Carboxyfluorescein succinimidyl ester (CFSE) (Biolegend) was dissolved in dimethyl sulfoxide (DMSO). Annexin V-APC was purchased from Immunotools. Anti-CD150PE and anti-GRPC5C Alexa Fluor 405 antibodies were purchased from R&D Systems. TGF-β1 was obtained from Peprotech.
7
Apoptosis Quantification by Flow Cytometry
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The 8 × 107 cells were plated in p60 dishes and the following day were treated with drugs. Following 72 h of treatment, the media were collected in 5 mL flow cytometry tubes and the cells were detached with Accutase (Euroclone, Milan, Italy). The cells were then washed with an annexin binding buffer (10 mM Hepes pH 7.4; 140 mM NaCl; 8 mM CaCl2) and stained with 3 µL of annexin V-APC (Immunotools, Friesoythe, Germany) and 1 µL of propidium iodide 0.1 mg/mL (Sigma Aldrich, Milan, Italy) in 100 µL of an annexin binding buffer for 30 min at RT. The cells were analyzed with BD FACSCanto II and analyzed using FlowJo software (BD Biosciences, distributed by DBA, Milan, Italy).
8
Evaluating Cell Viability: Annexin V and To-Pro-3 Staining
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Mec-1 cell viability was quantified with annexin V staining. Cells were collected and stained with annexin V-APC (Immunotools) in annexin V buffer (10-mM HEPES/NaOH, pH 7.5, 140-mM NaCl, and 2.5-mM CaCl2) for 15 min on ice in the dark. Samples were analyzed on a FACS Canto II flow cytometer. The viability of primary CLL cells was determined using the viability dye, To-Pro-3 (Molecular Probes), according to the manufacturer’s protocols. The samples were analyzed using the BD FACS Canto II flow cytometer (BD Bioscience, San Diego, CA, USA) by collecting 30,000 events. HS-5 cells were excluded from the analysis by gating out GFP+/FSChigh events. Statistical analysis was performed using FCSExpress (DeNovo Software Inc.,Pasadena CA, USA) and GraphPad Prism (GraphPad Software Inc., La Jolla, San Diego, CA, USA) software packages.
9
Real-Time Apoptosis and Necrosis Assay
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For real-time investigation of apoptosis and necrosis, cells were seeded at densities of 1 × 104 cells in 50 µl media into white 96-well plates and incubated for 24 hours at 37°C. Roginolisib (100 µM), paclitaxel (400 nM) or DMSO (0.1%) were added followed by immediate addition of Real Time-Glo Annexin V Apoptosis and Necrosis reagent (part# JA1011, Promega, Madison, WI, USA). Luminescence and fluorescence signals were monitored overtime up to 40 h using the EnSpire Multimode Plate Reader 2300 (PerkinElmer). For end-point investigation of apoptosis and necrosis, cells were seeded at densities of 1 × 105 cells in 1 ml media into 24-well plates and incubated for 24 hours at 37°C. Then, cells were treated with roginolisib (100 µM) or DMSO (0.1%) and incubated for a further 31 hours. After harvesting using accutase, cells were stained using Annexin V-APC (Immunotools, Friesoythe, Germany) and 25 µg/ml PI (propidium iodide) for 10 min. Cells were analysed by a BD FACSLyric Flow Cytometry System (BD, Heidelberg, Germany). Phase-contrast microscopy was used to investigate morphological changes. Cells (1 × 105 cells/2ml) were seeded into 6-well plates and incubated with roginolisib (100 µM) or DMSO for 24 h. Cells were imaged using a CKX41 inverted microscope and Olympus CellF (Olympus Life Science, Tokyo, Japan).
10
Cell Cycle and Apoptosis Analysis
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Cell cycle analysis was performed with Hoechst 33342, and flow cytometric measurements were performed using a Gallios flow cytometry instrument (Beckman Coulter, Inc.). Twenty-four hours after transfection, Jurkat T cells were collected, washed in 5 ml PBS and spun for 5 min at 200×g. Next, cells were resuspended in 0.5 ml PBS and incubated for 30 min with a 5 µg/ml final concentration of Hoechst 33342, which stains DNA in living cells. Samples were kept for 30 min at RT.
To analyze apoptosis, cells were labeled with fluorochromes Annexin V and 4′,6-diamidino-2-phenylindole (DAPI). Thus, viable (Annexin V−/DAPI−), early apoptotic (Annexin V+/DAPI−), late apoptotic (Annexin V+/DAPI+) and necrotic (Annexin V-/DAPI+) cells can be distinguished. Briefly, 24 h after transfection, cells were centrifuged (200×g, 5 min), washed with 5 ml of PBS and collected. Cells (1 × 106) were resuspended in 100 µl BB (Binding Buffer; 10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and incubated with 5 µl of Annexin V APC (Immunotools) for 15 min at RT in darkness. Next, 400 µl of BB and 3 µl of DAPI (1 mg/ml) were added to each sample. The acquisition of events was performed in a Gallios flow cytometry instrument (Beckman Coulter, Inc.) using specific software.
To analyze apoptosis, cells were labeled with fluorochromes Annexin V and 4′,6-diamidino-2-phenylindole (DAPI). Thus, viable (Annexin V−/DAPI−), early apoptotic (Annexin V+/DAPI−), late apoptotic (Annexin V+/DAPI+) and necrotic (Annexin V-/DAPI+) cells can be distinguished. Briefly, 24 h after transfection, cells were centrifuged (200×g, 5 min), washed with 5 ml of PBS and collected. Cells (1 × 106) were resuspended in 100 µl BB (Binding Buffer; 10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and incubated with 5 µl of Annexin V APC (Immunotools) for 15 min at RT in darkness. Next, 400 µl of BB and 3 µl of DAPI (1 mg/ml) were added to each sample. The acquisition of events was performed in a Gallios flow cytometry instrument (Beckman Coulter, Inc.) using specific software.
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