Cells were seeded in a 100 mm dish at a density of 2.4 × 105 cells/dish. The following day, cells were treated for 8 h with chrysin, E2, or chrysin + E2. After 8 h, the cells were fixed with 70% ethanol in PBS, and dead cells were stained with propidium iodide (PI). The cell cycle was examined using Novocyte2000 (Acea Biosciences, Inc., San Diego, CA, USA).
Novocyte 2000
Manufactured by Agilent Technologies
Sourced in United States
The Novocyte 2000 is a flow cytometer designed for advanced cell analysis. It offers high-performance flow cytometry with a compact footprint. The Novocyte 2000 provides reliable and consistent data for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase 2, by Worthington (3 mentions)
Alexa fluor488 ab184675, by Abcam (3 mentions)
Fitc annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Gelatin, by Merck Group (1 mentions)
Ab239686, by Abcam (1 mentions)
9 protocols using novocyte 2000
1
Chrysin and E2 Cell Cycle Analysis
2
Apoptosis Detection via Annexin-V/PI Staining
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Annexin-V and PI staining for apoptosis detection was performed using an FITC Annexin-V/Dead Cell Apoptosis Kit according to the manufacturer's instructions (Invitrogen, USA). Briefly, cells were treated with various concentrations of essential oils and incubated for 48 h. The cells were then collected by trypsinization, washed 2 times with cold PBS, suspended in 100 μL of a binding buffer (diluted from 10x binding buffer), and stained with 5 μL PI (50 μg/mL stock solution) and 5 μL FITC-labeled Annexin-V in the dark for 15 min at room temperature. The cells were analyzed by Novocyte 2000 (ACEA Biosciences Inc, USA). The percentages of Annexin-V + /PI− (apoptosis cells), Annexin-V/PI− (living cells), and Annexin-V + /PI+ (necrotic cells) staining were determined after marking for the positive and negative population.
3
Isolation and Characterization of Rat Pulmonary Artery Smooth Muscle Cells
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Female rat PASMC was isolated as described previously.23 (link) In brief, the whole lungs or the left lobe was excised from the rat and immediately placed in cold DMEM (Dulbecco’s Modified Eagle Medium) without glutamine and pyruvate (catalog No. 11960-044; Gibco). Under the hood, clean vessels from the lungs were cut and transferred to 2 mL of 1 mg/mL collagenase-2 (catalog No. LS004176; Worthington) in DMEM without glutamine and pyruvate for 20 minutes at 37 °C. The supernatant was aspirated carefully, and 10 mL of warm DMEM with glutamine supplemented (catalog No. 11965-092; Gibco) with 20% fetal bovine serum (catalog No. 25-5144; Geneclone) was added. This was then plated in a 10-cm dish covered with gelatin (catalog No. ES-006-B; Millipore). To ensure adequate cell number, cells were pooled from 5 different rats. Every 2 to 3 days, half of the media was changed to DMEM with glutamine. After 2 to 3 passages, the cells were stained for smooth muscle actin (anti-α-SM actin [1A4], Alexa-Fluor488 ab184675, Abcam, 1:100, isotype IgG2a) using FACS performed in a Novocyte 2000 (ACEA Biosciences) instrument.
4
Quantifying Intracellular ROS Levels
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To evaluate intracellular reactive oxygen species (ROS) levels by using the cell‐permeant H2‐DCFDA method, cells were treated with 10 μmol L−1 H2‐DCFDA for 30 minutes. Intracellular oxidative burst was then measured using a NovoCyte flow cytometer (NovoCyte 2000; ACEA Biosciences, San Diego, CA, USA), and data analysis was performed using NovoExpress Software.
5
Isolation and Characterization of Rat Pulmonary Artery Smooth Muscle Cells
6
Rat Pulmonary Artery Smooth Muscle Cell Isolation
7
Neuroprotective Effects of MJe Against 6-OHDA
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The protective effect of MJe against 6-OHDA-induced cell death was analyzed by fluorescence-activated cell sorting (FACS), using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining [21 (link)], a method employed to discriminate the living status of cells.
SH-SY5Y were seeded similarly to TB assay, but pretreated for 1 h with MJe (0.1 and 0.5 mg/mL) and then exposed to 50 μM 6-OHDA for another 24 h. Next, cells were collected and processed as already reported [21 (link)]. Finally, samples were run on a Novocyte 2000 cytofluorimeter (ACEA Biosciences Inc., San Diego, CA, USA).
Caspase 3 enzymatic activity was measured using a commercial kit (AbCam, Cambridge, UK). SH-SY5Y cells were differentiated in 100 mm petri dishes (1.5 × 106 cells) and then treated for 6 h, as explained above. Then, according to the manufacturer’s instructions, the analysis was carried out on cell lysates [17 (link)]. Absorbance was measured at 405 nm by a microplate spectrophotometer.
SH-SY5Y were seeded similarly to TB assay, but pretreated for 1 h with MJe (0.1 and 0.5 mg/mL) and then exposed to 50 μM 6-OHDA for another 24 h. Next, cells were collected and processed as already reported [21 (link)]. Finally, samples were run on a Novocyte 2000 cytofluorimeter (ACEA Biosciences Inc., San Diego, CA, USA).
Caspase 3 enzymatic activity was measured using a commercial kit (AbCam, Cambridge, UK). SH-SY5Y cells were differentiated in 100 mm petri dishes (1.5 × 106 cells) and then treated for 6 h, as explained above. Then, according to the manufacturer’s instructions, the analysis was carried out on cell lysates [17 (link)]. Absorbance was measured at 405 nm by a microplate spectrophotometer.
8
Exosome Characterization by Flow Cytometry
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The hBMMSC-Exos were characterized by their surface markers CD63 and CD81 using flow cytometry. First, exosomes were captured with CD63-conjugated capture beads (ab239686, abcam, USA) and labeled with phycoerythrin (PE)-conjugated CD81 antibody (130-118-481, Miltenyi, USA) after serum blockage. CD63 and CD81 positive exosomes were quantified using a flow cytometer (Novocyte 2000, Agilent, USA) against isotype control.
9
Evaluating ROS and Mitochondrial Potential
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Reactive oxygen species (ROS) and mitochondrial membrane potential (∆ψm) were evaluated by flow cytometry. HUVECs were plated on 6-well plates (8 × 104 cells/well) and treated as described for cell viability assays. Each sample was harvested, centrifuged at 1200 rpm for 5 min, washed with PBS, and resuspended in a serum-free medium. Aliquots of cell suspension were loaded with 10 µM DCFH-DA for 30 min at 37 °C and used to evaluate the intracellular ROS [30 (link)]. The signals of the highly fluorescent oxidized derivative 2′-7′-dichlorofluorescein (DCF), formed in the presence of ROS, were collected with Novocyte 2000 cytofluorimeter (Agilent, Santa Clara, CA, USA). To assess the trans-membrane potential (Δψm), we stained the HUVECs cells with 10 μM rhodamine 123 (R123, Invitrogen Molecular Probes), a fluorescent probe that accumulates in the matrix of functional mitochondria, for 10 min at 37 °C [31 (link)]. Signals were collected cytofluorimetrically.
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