To detect synergistic effects of the chemotherapeutic drugs on cell apoptosis, treated cells were analyzed using the fluorescein isothiocyanate-Annexin V apoptosis kit (BD Biosciences, San Jose, CA, USA). The results were analyzed by flow cytometry using an FACSAria III device (BD Biosciences). All the experiments were repeated independently in triplicate.
Facsaria 3 device
Manufactured by BD
Sourced in United States
The FACSAria III is a cell sorter device manufactured by BD Biosciences. The core function of the FACSAria III is to rapidly analyze and physically separate complex heterogeneous populations of cells based on their specific cellular characteristics, such as size, granularity, and the expression of fluorescently labeled markers.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fluorescein isothiocyanate apoptosis kit, by BD (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
Anti pe beads, by Miltenyi Biotec (1 mentions)
Optiprep gradient solution, by Merck Group (1 mentions)
Egta solution, by Merck Group (1 mentions)
Accutase, by STEMCELL (1 mentions)
Facsdiva 8, by BD (1 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
5 protocols using facsaria 3 device
1
Detecting Synergistic Apoptosis Effects
2
Isolation of Liver Non-Parenchymal Cells
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Liver non-parenchymal cells were isolated as previously described44 (link),45 (link). Briefly, the liver was washed with EGTA solution (Sigma Aldrich, St. Louis, MO) and digested with type 1 collagenase (Worthington Biochemical Corporation, Lakewood, NJ) through the portal vein. Hepatic non-parenchymal cells were separated from hepatocytes by centrifugation. HSCs were separated from non-parenchymal cells using Optiprep gradient solution (Sigma-Aldrich, St. Louis, MO). Monocytes and Kupffer cells were isolated using the FACS Aria III device (BD Bioscience, San Jose, CA) and liver sinusoidal endothelial cells (LSECs) were separated using magnetic-activated cell sorting (MACS) with anti-CD146-PE and anti-PE beads (Miltenyi Biotec, San Diego, CA) following manufacturer’s protocol.
3
Multiparametric Flow Cytometry of Murine Hematopoietic Cells
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BM mononuclear cells were stained on ice in 50 µL volume with a mixture of labeled antibodies for 90 min. Cell-sorting and flow-cytometric analysis were performed using the FACS-Aria III device (BD). Specific antibodies from Biolegend (San Diego, CA, USA): Lineage cocktail-Pacific Blue, cKit-APC-Cy7, Sca1-APC, CD45.2–Pacific Blue, Gr-1 (Ly-6G/Ly-6C)-FITC, Ter119-PerCP/Cy 5.5, CD41-PE and CD150-PE-Cy7; eBiosccience (San Diego, CA, USA): CD34-FITC, and FcγR-PE; TONBO Biosciences (San Diego, CA, USA): CD45.1-APC and Mac-1 (CD11b)-PE-Cy7.
4
Isolation of Dopaminergic Neurons
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Dopaminergic neuronal cultures differentiated from parental and TH-mCherry reporter iPSCs were harvested 50 days after starting the differentiation regime. Briefly, cells were washed with 1x PBS, detached using Accutase (Stemcell Technologies) and singularized by filtering through a 37 μm reversible cell strainer (Stemcell Technologies). Subsequently, cells were pelleted, resuspended in 1 ml of 1x PBS, and sorted on a BD FACSAria™ III device (Becton, Dickinson and Company) to obtain mCherry-positive and mCherry-negative fractions.
5
Characterization of Adipose-Derived MSCs
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Adipose tissue-derived MSCs were obtained from the American Type Culture Collection (ATCC PCS-500-011) and were grown in DMEM containing 10% FBS (Fetal Bovine Serum), 4 mM L-glutamine, 100 U/mL penicillin-streptomycin, and 5 ng/mL bFGF. Cells were expanded until the 5th in vitro passage for proteome analysis and production of Muse and non-Muse cells (see below).
Flow cytometric characterization was performed by labeling cells with the following primary antibodies: CD34-PE, CD45-FITC, CD44-PE, CD73-APC, CD90-FITC, and CD105-PerCpCy5. The antibodies were used according to the manufacturer’s procedures (Santa Cruz Biotechnology, Dallas, TX, USA). After 30 min of incubation with the antibodies at room temperature, cells were washed with PBS and resuspended in FACS (Fluorescence Activated Cell Sorting) buffer for data acquisition on BD FACS Aria III device, and BD FACS Diva 8.0.1 program (Becton, Dickinson, Franklin Lakes, NJ, USA).
Flow cytometric characterization was performed by labeling cells with the following primary antibodies: CD34-PE, CD45-FITC, CD44-PE, CD73-APC, CD90-FITC, and CD105-PerCpCy5. The antibodies were used according to the manufacturer’s procedures (Santa Cruz Biotechnology, Dallas, TX, USA). After 30 min of incubation with the antibodies at room temperature, cells were washed with PBS and resuspended in FACS (Fluorescence Activated Cell Sorting) buffer for data acquisition on BD FACS Aria III device, and BD FACS Diva 8.0.1 program (Becton, Dickinson, Franklin Lakes, NJ, USA).
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