For primary AML samples, cells were stained with anti-human CD45 conjugated to allophycocyanin-cyanine (APC-H7; 2D1, BD Biosciences, Franklin Lakes, NJ, USA), CD34 conjugated to phycoerythrin-cyanine-7 (PECy7; 581, BD Biosciences, Franklin Lakes, NJ, USA ) and CD38 conjugated to allophycocyanin (APC; HB7, BD Biosciences, Franklin Lakes, NJ, USA ) and Ferroportin (FPN) conjugated to Alexa Fluor-405 (AF-405; 8G10NB, Novus Biologicals, Littleton, CO, USA) or isotype control AF-405 (mouse IgG2b; MPC11 (Novus Biologicals, Littleton, CO, USA )) for 30 min in the dark at 4oC. For cell lines, cells were only stained with FPN-AF-405 or Isotype control for 30 min in the dark at 4oC. Cells were washed two times and resuspended in cold FACS buffer containing a viability dye (7-aminoactinomycin; 7AAD, Life Technologies, Carlsbad, CA, USA). Cells were evaluated in a BD-LSRII analytical flow cytometer. Analysis was later performed using FlowJo 9.3 software for Mac OS X (TreeStar, Ashland, OR).
7 amino actinomycin
Manufactured by Thermo Fisher Scientific
Sourced in United States
7-amino-actinomycin is a fluorescent dye used in flow cytometry applications to measure cell viability and apoptosis. It selectively binds to DNA, emitting a red fluorescence when excited, allowing the identification of dead or dying cells.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 phycoerythrin, by Thermo Fisher Scientific (3 mentions)
Facsaria 2, by BD (3 mentions)
Apc h7 2d1, by BD (2 mentions)
Pecy7 581, by BD (2 mentions)
Apc hb7, by BD (2 mentions)
Af 405 8g10nb, by Novus Biologicals (2 mentions)
Mouse igg2b mpc11, by Novus Biologicals (2 mentions)
Lsrii analytical flow cytometer, by BD (2 mentions)
Anti cd8 rpa t8, by BD (1 mentions)
Magnisort human t cell enrichment kit, by Thermo Fisher Scientific (1 mentions)
Surface antibodies, by Thermo Fisher Scientific (1 mentions)
Annexin 5 binding buffer, by BD (1 mentions)
Allophycocyanin conjugated annexin 5, by BD (1 mentions)
Pharm lyse lysing buffer, by BD (1 mentions)
8 protocols using 7 amino actinomycin
1
Ferroportin Expression in Primary AML
2
Ferroportin Expression in Primary AML
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For primary AML samples, cells were stained with anti-human CD45 conjugated to allophycocyanin-cyanine (APC-H7; 2D1, BD Biosciences, Franklin Lakes, NJ, USA), CD34 conjugated to phycoerythrin-cyanine-7 (PECy7; 581, BD Biosciences, Franklin Lakes, NJ, USA ) and CD38 conjugated to allophycocyanin (APC; HB7, BD Biosciences, Franklin Lakes, NJ, USA ) and Ferroportin (FPN) conjugated to Alexa Fluor-405 (AF-405; 8G10NB, Novus Biologicals, Littleton, CO, USA) or isotype control AF-405 (mouse IgG2b; MPC11 (Novus Biologicals, Littleton, CO, USA )) for 30 min in the dark at 4oC. For cell lines, cells were only stained with FPN-AF-405 or Isotype control for 30 min in the dark at 4oC. Cells were washed two times and resuspended in cold FACS buffer containing a viability dye (7-aminoactinomycin; 7AAD, Life Technologies, Carlsbad, CA, USA). Cells were evaluated in a BD-LSRII analytical flow cytometer. Analysis was later performed using FlowJo 9.3 software for Mac OS X (TreeStar, Ashland, OR).
3
Apoptosis Analysis in MCF-7 Cells
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Apoptosis in the siRNA-treated MCF-7 cells was analysed using flow cytometry (FACSAria II, BD Biosciences). The cultured cells were washed twice with PBS and were resuspended in binding buffer at a concentration of 1 × 106 cells/ml. The cell suspensions (1 × 105 cells/100 μl) were transferred into 5 ml culture tubes, and 5 μl of annexin V-phycoerythrin (eBioscience) and 5 μl of 7-amino-actinomycin (eBioscience) were then added. The cells were gently vortexed and incubated at room temperature in the dark for 15 min. Subsequently, another 400 μl of aliquot of binding buffer were added. Flow cytometry was performed within 4 h of staining.
4
Isolation and Activation of Human T Cells
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Human T-cells were purified from frozen buffy coats and isolated by negative selection using the MagniSort Human T Cell Enrichment Kit (Thermofisher). T cells were seeded into wells of a 48 well plate at a density of 1 × 106 cells/well and activated at a 1:1 ratio with Human T activator CD3/CD28 conjugated Dynabeads (ThermoFisher). Cells were cultured in a humidified CO2 incubator at 37 °C for 6 days in either organoid media or RPMI plus 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 10 mM Hepes buffer, 69 mM L-arginine and 50 μg beta-mercaptoethanol. After 6 days in culture, cells were stained with fluorochrome-conjugated monoclonal antibodies: anti-CD3 (SK7), anti-CD4 (OKT-4) and anti-CD62L (DREG-56) were obtained from eBioscience; and anti-CD8 (RPA-T8) was obtained from BD Biosciences. Cells were stained with 7-aminoactinomycin (7AAD, eBioscience) as a viability marker. Flow cytometry was performed on a LSRII flow cytometer. (BD Biosciences), and the data were analyzed using FlowJo software version 10 (FlowJo, LLC).
5
Apoptosis Assay with GM-CSF
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Cells cultured for 24 h with (Eca109/GM and 9706/GM) or without (Eca109 and 9706) rhGM-CSF were washed twice with PBS and then re-suspended in 1× working solution at 1×106 cells/mL. A total of 100 μL of cell suspension was incubated with 5 μL of annexin v-phycoerythrin (fluorescein isothiocyanate; eBioscience, San Diego, CA, USA) and 5 μL of 7-amino-actinomycin (propidium iodide; eBioscience) in the dark for 20 min. After staining, 400 μL of binding buffer was added and data were acquired by flow cytometry within 4 h of staining.
6
Apoptosis Analysis in MCF-7 Cells
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Apoptosis in the siRNA-treated MCF-7 cells was analyzed using flow cytometry (FACSAria II, BD Biosciences). The cultured cells were washed twice with PBS and were resuspended in binding buffer at a concentration of 1 × 106 cells/mL. The cell suspensions (1 × 105 cells/100 µL) were transferred into 5-mL culture tubes, and 5 µL of annexin V-phycoerythrin (eBioscience) and 5 µL of 7-amino-actinomycin (eBioscience) were then added. The cells were gently vortexed and incubated at room temperature in the dark for 15 min. Subsequently, another 400-µL aliquot of binding buffer was added. Flow cytometry was performed within 4 h of staining.
7
Apoptosis Assay for Anti-PADI2 siRNA
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MNK-45 cells or Bel-7402 cells with the treatment of anti-PADI2 siRNA were washed twice with PBS and were resuspended in binding buffer at a particular concentration. The cell suspensions at a concentration of 1×105 cells/100 μL were transferred into 5 mL culture tubes, and 5 μL of annexin V-phycoerythrin (eBioscience, San Diego, CA, USA) and 5 μL of 7-amino-actinomycin (eBioscience) were added to the tube. The mixture was placed at room temperature in the dark for 15 min. Later, another 400 μL aliquot of binding buffer was added. The apoptosis ratio was examined by flow cytometry (FACSAria II; BD Biosciences, Franklin Lakes, NJ, USA).
8
Murine Peripheral Blood Flow Cytometry
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For murine peripheral blood flow cytometry analysis, peripheral blood was collected by retro-orbital bleeding and incubated with surface antibodies (eBioscience) in staining buffer (phosphate-buffered saline (PBS) supplemented with 0.5% FBS). Peripheral blood cell suspensions were treated with Pharm Lyse Lysing Buffer (BD Biosciences) and washed twice with PBS. For apoptosis assays, cells were incubated with allophycocyanin-conjugated Annexin V (BD Bioscience) for 15 min at RT in 1X Annexin V Binding Buffer (BD Bioscience) following staining with 7-aminoactinomycin (eBioscience). Data were acquired on a FACSCanto I and results were analyzed using FlowJo Version 10 (FlowJo). The list of antibodies used for flow cytometry analysis of mouse and human cells is indexed in Supplementary Data 4 .
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