The human glioma stem/progenitor cell line SU3 and nude mice expressing EGFP (NC-CB57/6J-EGFP) were prepared by our laboratory [4 (link), 5 (link)]. The remaining reagents were purchased from companies as follows: rat C6 glioma cell line (Shanghai Institutes for Biological Sciences), RFP transgenic kit (Genechem, Shanghai), γ-secretase inhibitors DAPT (GSI-IX) (Selleck), Dulbecco's Modified Eagle's Medium (Gibco), fetal bovine serum (Hyclone), Caspase—Glo 3/7 assay kit (Promega), Trizol solution (Invitrogen), reverse transcription kit (Fermentas), ECL chemiluminescence reagent, trypan blue, GAPDH antibody (Biyuntian, Shanghai), Notch-1, NICD, Bcl-2 and pAKT antibodies (Cell Signal), 2', 3'-cyclic nucleotide 3' phosphodiesterase (CNP) monoclonal antibody (Abcam), qPCR apparatus and SYBR Green qPCR Mix (BioRad), apoptosis kit (BD), flow cytometry instrument (Beckman), inverted fluorescence microscope (Carl Zeiss), linear accelerator (Siemens Primus), and in vivo fluorescence imaging system (Maestro EX, CRi).
Flow cytometry instrument
Manufactured by Beckman Coulter
Sourced in United States
The flow cytometry instrument is a laboratory equipment used to analyze and sort cells or particles suspended in a fluid stream. It measures and analyzes multiple physical characteristics of cells or particles as they flow through a laser beam.
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Lab products found in correlation
Reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Sybr green qpcr mix, by Bio-Rad (1 mentions)
Inverted fluorescence microscope, by Zeiss (1 mentions)
Trizol solution, by Thermo Fisher Scientific (1 mentions)
Dapt gsi 9, by Selleck Chemicals (1 mentions)
Ecl chemiluminescence reagent, by Beyotime (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Caspase glo 3 7 assay kit, by Promega (1 mentions)
Annexin 5 fluos staining kit, by Roche (1 mentions)
Annexin 5 fitc propidium iodide apoptosis detection kit, by Vazyme (1 mentions)
Propidium iodide solution, by Beyotime (1 mentions)
6 protocols using flow cytometry instrument
1
Glioma Stem Cell and Xenograft Model
2
Apoptosis Analysis of Tamoxifen and Estradiol
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DLD-1 cells (~5×105) were seeded onto 60-mm tissue culture plates and cultured for 24 h. The culture medium was then discarded and fresh culture medium containing TAM or E2 was added. DMSO was used as a control. The experimental design was based on the MTT assay results. The concentration of drug that exhibited a 30% reduction in cell viability (IC30), which demonstrated a relatively low inhibitory effect, and the IC70, which demonstrated a greater inhibitory effect, were used as a basis for grouping. Cells were treated with the following: DMSO (control); 0.0625×10−3 M E2; 0.5×10−3 M E2; 0.0625×10−4 M TAM; 0.25×10−4 M TAM; 0.0625×10−3 M E2 + 0.0625×10−4 M TAM; 0.5×10−3 M E2 + 0.25×10−4 M TAM. The cells were cultured for 24, 48 or 72 h, then the supernatant was collected and adherent cells were dissociated with trypsin by centrifuging at 500 × g for 5 min and 4°C for downstream analysis. The cells were then washed with phosphate-buffered saline (PBS), centrifuged again at 500 × g for 5 min and 4°C and double-stained using the Annexin-V-FLUOS Staining kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions in the dark for 5 min. Apoptosis of the cells was measured using a flow cytometry instrument (Beckman Coulter, Inc., Brea, CA, US).
3
Apoptosis Measurement of Granulosa Cells
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Annexin V-fluorescein isothiocyanate (FITC) staining reagent (Vazyme, China) was used to measure the apoptosis of granulosa cells. Briefly, the cells were washed three times with cold PBS by centrifugation at 300 g for 5 min. The cell pellet was resuspended in 100 μL of binding buffer containing 5 μL of Annexin V-FITC and 5 μL of PI staining solution at a density of 1 × 106/mL, and incubated for 10 min at room temperature in the dark. Then, 400 μL of binding buffer was added to the stained cell suspension. The stained cells were detected within 1 h by a flow cytometry instrument (Beckman Coulter, Brea, CA, USA).
4
Apoptosis Induction in Cell Cultures
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The cells were seeded into 6-well culture plates at a density of 1 × 10 6/mL cells per well. The cells were treated with 0.5 μM U73122 or m3M3FBS for 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h, respectively, and then the cells were washed three times with cold PBS by centrifugation at 800 g for 5 min. Cell apoptosis was assessed with an annexin V-FITC/propidium iodide apoptosis detection kit (Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions, with the cell pellet resuspended in 100 μL of 1 × binding buffer and adjusted to the density to 1 × 10 6/mL. The stain was detected within 1 h by a flow cytometry instrument (Beckman Coulter, CA, USA).
5
Cell Cycle Analysis by Flow Cytometry
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To evaluate cell cycle, flow cytometry was performed 48 h after transfection. hCM cells were seeded in six‐well plates. Cells were fixed in 70% cold ethanol and stained with propidium iodide solution (Beyotime). All labeled cells were analyzed on a flow cytometry instrument (Beckman Coulter, Fullerton, CA, USA).
6
Measurement of Intracellular ROS in VSMCs
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The VSMCs (5 × 105 cells/dish) were seeded in DMEM containing 10% FBS in 6-cm culture dishes, and grown until the monolayer was confluent. The cells were treated with DCF-DA (20 μM) for 20 min. After treatment with PMC (20 and 50 μM) or an equal volume of solvent control (0.1% DMSO) for 20 min., cells were stimulated with PDGF-BB (10 ng/ml or LPS/IFN-γ. The cells were washed with PBS before trypsinization. The levels of intracellular ROS were detected using a flow cytometry instrument (Beckman Coulter, Brea, CA, USA). Data were collected from 10,000 cells per experimental group. All experiments were repeated at least three times to ensure reproducibility.
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