EPs were labeled with fluorescent (flash red) SPM particles (0.9 µm diameter; Bangs Laboratories, Fishers, IN, USA) by incubation in cell culture for 24 h. Successful labeling was confirmed by flash-red fluorescence. Labeling efficiency was assessed by flow cytometry. In vitro toxicity experiments were performed 24 h after SPM labeling. Cell viability was assessed by trypan blue exclusion. Apoptosis and necrosis were assessed by flow cytometry using 7AAD and annexin-V stains (BD Pharmingen, San Jose, CA, USA). Albumin (Alb) and α-fetoprotein messenger RNA (mRNA) abundance were measured by quantitative real-time polymerase chaing reaction (PCR) (see below).
7aad and annexin 5
Manufactured by BD
Sourced in United States
7AAD and annexin-V are fluorescent dyes used in flow cytometry applications. 7AAD is a DNA-binding dye that can be used to detect late-stage apoptosis or cell death. Annexin-V is a protein that binds to phosphatidylserine, which is exposed on the surface of cells undergoing apoptosis. Together, these two markers can be used to identify and quantify different stages of cell death.
Automatically generated - may contain errors
Lab products found in correlation
Diethyl ether, by Avantor (1 mentions)
Trifluoroacetic acid, by Avantor (1 mentions)
Ninhydrin, by Merck Group (1 mentions)
Methanol, by Avantor (1 mentions)
Fmoc amino acids, by AAPPTec (1 mentions)
4 methylpiperidine, by Merck Group (1 mentions)
Calcium assay kit, by BD (1 mentions)
N n dicyclohexylcarbodiimide, by AAPPTec (1 mentions)
N n diisopropylethylamine dipea triisopropylsilane tips 1 2 ethanedithiol edt, by Merck Group (1 mentions)
6 chloro 1 hydroxy benzotriazole 6 cl hobt, by AAPPTec (1 mentions)
N n dimethylformamide (dmf), by Avantor (1 mentions)
Pyridine, by Merck Group (1 mentions)
Rink amide resin, by AAPPTec (1 mentions)
Flow cytometry, by BD (1 mentions)
Fluorescein di β d galactopyranoside fdg, by Merck Group (1 mentions)
Anti mouse f4 80 bm8, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
4 protocols using 7aad and annexin 5
1
Evaluating Nanoparticle Toxicity on Cells
2
Cell Viability and Apoptosis Assay Protocol
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MCF-7 and CRL-3271 cells were obtained from ATCC (Manassas, VA, USA), fetal bovine serum (FBS) was obtained of Gibco. MitoProbe™ JC-1 Assay Kit (Termofisher; M34152), Calcium Assay Kit (BD Biosciences; 640176), 7AAD and Annexin V (BD Biosciences; 559763) N,N-diisopropylethylamine (DIPEA), triisopropylsilane (TIPS), 1,2-ethanedithiol (EDT), 4-methylpiperidine, pyridine, and ninhydrin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rink amide resin, Fmoc-amino acids, 6-chloro-1-hydroxy-benzotriazole (6-Cl–HOBt), and N,N-dicyclohexylcarbodiimide (DCC) were purchased from AAPPTec (Louisville, KY, USA). Methanol, diethyl ether, N,N-dimethylformamide (DMF), absolute ethanol, dichloromethane (DCM), acetonitrile (ACN), isopropylalcohol (IPA), and trifluoroacetic acid (TFA) were obtained from Honeywell-Burdick & Jackson (Muskegon, MI, USA). All reagents were used without further purification.
3
Apoptosis and Cell Cycle Analysis
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Flow cytometry (BD) was employed for the apoptosis and cell cycle assays. To be specific, FLS-RA (1.5 ∗ 105 cells/well) were seeded into the 6-well plates and treated with berberine (12.5 and 25 μM) for 24 h. Then, cells were digested with EDTA-free trypsin, and a portion of cells were stained with 7-AAD and Annexin V (BD, 559763) for 30 min in dark at room temperature for apoptosis assay; meanwhile, the other portion of cells were stained with PI for cell cycle assay in accordance with the manufacturer instructions. The FlowJo software was used for analyses.
4
Macrophage Activation and Viability Assay
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BMDMs were detached from Petri dishes by a 20 min-treatment at 4ºC in the presence of PBS 2% (v/v) iFBS, 5mM EDTA. Cells were subsequently subjected to two independent flow cytometry analyses: i) to check for viability [7-AAD and annexin V (BD Biosciences)], for the macrophage surface marker F4/80 [anti-mouse F4/80 (BM8) (Biolegend)], as well as activation markers [MHC class II (M5/114.15.2), CD40 (3/23) and CD80 (16-10A1) (Biolegend)]; and ii) to measure β-glucocerebrosidase activity using fluorescein di-β-D-galactopyranoside (FDG, Sigma). Unstained cells were used as negative control. Cells were acquired in a FACS Canto II (BD Biosciences) using the BD FACSDiva™ software (BD Biosciences). Data analysis was performed with FlowJo® v10 (BD Biosciences).
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