For ferroptosis analysis, BMCs were firstly stained with HSC markers and carefully washed. Cell death analysis was performed by suspending cells in a 7-amino-actinomycin D (7-AAD) staining solution (eBioscience) and incubating for 15 min at room temperature. Lipid peroxidation was measured by suspending cells in prewarmed (37 °C) PBS with 10 μM Liperfluo (Dojindo Molecular Technologies, Kumamoto, Japan) and incubating for 30 min at 37 °C. Ferrous ion deposition was measured by suspending cells in prewarmed (37 °C) PBS with 2 μM FerroOrange (Dojindo Molecular Technologies) and incubating for 30 min at 37 °C. For detection of 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), cells were fixed with IC Fixation buffer (eBioscience) at room temperature for 30 min. Then, cells were permeabilized with Permeabilization buffer (eBioscience) in the presence of anti-4-HNE or anti-MDA antibodies (all Abcam) at room temperature for 45 min and stained with fluorescent dye conjugated secondary antibodies (Thermo Fisher Scientific) at room temperature for another 30 min and finally analyzed by flow cytometry. 4-HNE and MDA levels LT-HSCs were also respectively determined using 4-HNE and MDA ELISA Kits (all Elabscience, Texas, USA) according to the manufacturers’ protocols.
7 amino actinomycin d 7 aad staining solution
Manufactured by Thermo Fisher Scientific
7-amino-actinomycin D (7-AAD) staining solution is a fluorescent dye used for DNA staining in flow cytometry analysis. It binds to DNA and emits fluorescence upon excitation, allowing the detection and quantification of cellular DNA content.
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Lab products found in correlation
Ferroorange, by Dojindo Laboratories (2 mentions)
Liperfluo, by Dojindo Laboratories (2 mentions)
Fluorescent dye conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Anti cd45, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Anti vegfr2, by R&D Systems (1 mentions)
Anti cd31, by BD (1 mentions)
Anti hla dr, by BD (1 mentions)
Anti cd14, by BD (1 mentions)
Anti cd68, by BD (1 mentions)
Mouse igg1, by R&D Systems (1 mentions)
Facscanto, by BD (1 mentions)
4 protocols using 7 amino actinomycin d 7 aad staining solution
1
Ferroptosis Analysis in Hematopoietic Stem Cells
2
Ferroptosis Analysis in BMCs
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For ferroptosis analysis, BMCs were firstly stained with HSC markers and carefully washed. Cell death analysis was performed by suspending cells in a 7-amino-actinomycin D (7-AAD) staining solution (eBioscience) and incubating for 15 min at room temperature. Lipid peroxidation was measured by suspending cells in prewarmed (37 °C) PBS with 10 µM Liperfluo (Dojindo Molecular Technologies, Kumamoto, Japan) and incubating for 30 min at 37 °C. Ferrous ion deposition was measured by suspending cells in prewarmed (37 °C) PBS with 2 µM FerroOrange (Dojindo Molecular Technologies) and incubating for 30 min at 37 °C.
3
Apoptosis Analysis of Hematopoietic Stem Cells
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HSC apoptosis was analyzed using an Annexin V-FITC Apoptosis Detection Kit (eBioscience) according to the manufacturer’s instructions. Briefly, BMCs were firstly stained with surface markers for HSCs and carefully washed. BMCs were then resuspended in 1 mL of 1× binding buffer. Then, annexin V-FITC antibody was added and incubated at room temperature for 10 min. After washing with 1× binding buffer for twice, a 7-amino-actinomycin D (7-AAD) staining solution (eBioscience) was added and immediately analyzed by a FACSverse flow cytometer. Apoptotic cells were identified as annexin V-positive cells.
4
Characterizing Macrophage Differentiation
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Differentiation of MACs in response to infection (MOI 300) was assessed via flow cytometry. Detached cells were fixed in 1% PFA, blocked with 10% FCS and incubated with the following monoclonal murine human-specific Abs for surface antigens: anti-CD14, anti-CD45, anti-HLA-DR, anti-CD31 (Becton Dickinson) and anti-VEGF-R2 (R&D Systems). For detection of intracellular antigens, cells were permeabilized with Cytofix Cytoperm and incubated with anti-CD68 (Becton Dickinson). In each experiment, control groups were stained with immunoglobulin isotype control Abs [mouse IgG2aκ, IgG1κ, IgG2bκ, (Becton Dickinson) and mouse IgG1 (R&D Systems)]. For assessment of cell viability, MACs isolated from Matrigel structures were incubated with 7-amino-actinomycin D (7AAD) staining solution (eBiosciences) and fixated with 1% PFA. Unstained cells were used as negative controls. Cells were analysed on a fluorescence-activated cell sorter (FACS Canto, Becton Dickinson) and results were analysed using Flowing Software (Terho, 2012) .
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