Mononuclear cells (1 × 106 per 100 μl) from cord blood were stained with the following antibodies (all from BD Biosciences, unless stated otherwise; the dilution used and catalogue numbers are in parentheses): anti-CD45RA–FITC (1:25; 555488), anti-CD90–APC (1:50; 561971), anti-CD135–Biotin (1:10; 624008), anti-CD38–PE-Cy7 (1:200; 335790), anti-CD10–Alexa Fluor 700 (1:10; 624040), anti-CD7–Pacific Blue (1:50; 642916), anti-CD45–V500 (1:200; 560777), anti-CD34–APC-Cy7 (1:100), anti-CD34–PerCP-Efluor 710 (1:100; eBioscience, 46-0344-42), anti-CD33–PE-Cy5 (1:100; Beckman Coulter, PNIM2647U), anti-CD19–PE (1:200), anti-CD3–FITC (1:100; 349201), anti-CD56–Alexa Fluor 647 (1:100; 557711) and Streptavidin–QD605 (1:200; Invitrogen, Q10101MP). Cells were sorted on a FACS Aria III system (BD Bioscience).
Facs aria 3 system
Manufactured by BD
Sourced in United States
The FACS Aria III system is a high-performance cell sorter designed for flow cytometry applications. It is capable of sorting multiple cell populations simultaneously with high purity and yield. The system features advanced optics, fluidics, and electronics to ensure reliable and accurate cell sorting.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 1, by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by Merck Group (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Verapamil, by Merck Group (2 mentions)
Halotag tmr ligand, by Promega (2 mentions)
Percp cy5, by BD (2 mentions)
Bv421 conjugated anti mouse cd4 rat monoclonal antibody rm4 5, by BD (2 mentions)
Pe conjugated anti mouse ifn γ rat monoclonal antibody xmg1.2, by BD (2 mentions)
Apc or pe conjugated anti mouse il 17a rat monoclonal antibody tc11 18h10, by BD (2 mentions)
Macs system, by Miltenyi Biotec (2 mentions)
Cytofix cytoperm reagent, by BD (2 mentions)
Facsaria 3, by BD (2 mentions)
Anti cd133, by Miltenyi Biotec (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rnase a, by Merck Group (2 mentions)
Immunoglobulin g igg isotype antibody, by Miltenyi Biotec (2 mentions)
2 7 dichlorofluorescein dcfh da, by Beyotime (2 mentions)
Vwf pe, by R&D Systems (2 mentions)
Ve cadherin fitc, by Thermo Fisher Scientific (2 mentions)
75 protocols using facs aria 3 system
1
Cord Blood Mononuclear Cell Phenotyping
2
Identification and Characterization of Innate Lymphoid Cells
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Mononuclear cells were isolated from SF using density gradient centrifugation with LSM (MP Biomedicals, LLC, Santa Ana, CA, USA). ILCs were defined as single cells within the lymphocyte gate on the scatter plot that were CD45 positive, lineage (CD3, CD19, CD16, CD94, CD14, CD1c, CD11b, CD11c, CD235ab, FcεRI, CD34) negative, and CD127 (IL-7R) positive. ILC subsets were classified based on the expression of CD117 (c-Kit), chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2) (CD294), and NKp44 (CD336). Specifically, ILC2s were defined as CRTH2 positive, and other subsets were CRTH2 negative. ILC1s, NKp44− ILC3s, and NKp44+ ILC3s were defined as CD117− NKp44−, CD117+ NKp44−, and CD117+ NKp44+, respectively. CCR6+ ILCs were defined as CCR6-positive cells in total ILCs. Antibodies used in flow cytometric analysis are listed in Additional file 7 : Table S1 and were purchased from BD Biosciences (San Jose, CA, USA), BioLegend (San Diego, CA, USA), Beckman Coulter (Villepinte, France), or MD Bioproducts (Saint Paul, MN, USA). Cells were analyzed with a FACS Aria III system (BD Biosciences). FlowJo software (Tree Star Inc., Ashland, OR, USA) was used to analyze flow cytometric data.
3
Cell Cycle and Mitochondrial Dynamics Assay
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Flow cytometric analysis was used to measure the cell cycle state of Jurkat T cells exposed to NOC, 2-MeO-E2, or CPT in the absence or presence of CMEP-NQ and was performed using a FACS Calibur (BD Science, San Jose, CA, USA) as described elsewhere [26 (link)]. The changes in the mitochondrial membrane potential (Δψm) following treatment with 0.3 μM NOC, 1.0 μM 2-MeO-E2, or 0.02 μM CPT in the absence or presence of CMEP-NQ were measured after staining with DiOC6 [27 (link),29 (link)]. Activation of BAK in Jurkat T cells following the same treatments was measured as described previously [30 (link)]. To measure intracellular ROS level, the cells were treated with DHE at 37°C for 30 min, and the mean fluorescent intensity (MFI) was analyzed using flow cytometry (FACS Aria III system, BD Science, USA) at an excitation wavelength of 488 nm.
4
CRISPR/Cas9-Mediated EDEM3 Knockout in HEK293T Cells
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HEK293T cells were co-transfected with CRISPR/Cas9 (sc-408476) and HDR (sc-408476-HDR) plasmid system from Santa Cruz Biotechnology (Dallas, TX, USA), following provider’s guidelines. At 48 h post transfection, 4 µg/mL puromycin was added to the cell media for clone selection. The cells were maintained in the supplemented media for three passages and further, the puromycin concentration was dropped to 2 µg/mL. Selection efficiency was verified by expression level of EDEM3 by Western blotting. The cells were sorted using a FACS Aria III system (BD Biosciences, San Jose, CA, USA). EDEM3 expression level was tested again by Western blotting and the clone presenting no expression of EDEM3 was further used.
5
Quantifying Cancer Stem Cells via Flow Cytometry
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For SP analyses, cells after treatment were digested with trypsin, washed with phosphate-buffered saline, and then resuspended in Dulbecco’s modified Eagle’s medium/F-12 medium (Gibco-BRL) containing 2% FBS at a density of 1 × 106 cells/mL, and stained with 5 μg/mL Hoechst 33342 (Sigma-Aldrich) in the presence or absence of 50 μM verapamil (Sigma-Aldrich) at 37 °C for 90 min. At the end of the incubation period, cells were counterstained with 2 μg/mL propidium iodide (Sigma-Aldrich). Analyses were performed using a FACS AriaIII system (BD Biosciences, San Jose, CA, USA). For CD44+/EpCAM+ cell analyses, cells after treatment were washed, resuspended, and then incubated at 4 °C in the dark for 40 min with fluorescence-conjugated monoclonal antibodies obtained from BD Biosciences against human CD44-FITC and EpCAM-Percp-Cy5.5, and their isotype IgG1 at concentrations recommended by the manufacturer. The samples were analyzed on a FACS Aria III (BD Biosciences). The results were analyzed using FlowJo software (Ashland, OR, USA). All FACS plots in our results have their corresponding negative controls. We have listed the complete FACS plots in supplementary materials .
6
Inducible Suicide Gene in hiPSCs
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253G1‐hiPSCs 43 (provided by Prof. Shinya Yamanaka at CiRA, Kyoto University) were transduced with the Tet‐inducible HSVtk lentiviral vector at a multiplicity of infection (MOI) of 2–10. Almost 100% transduction efficiency was observed based on examining humanized Kusabira‐Orange 1 fluorescent protein (hKO1) 44 expression under a fluorescence microscope. Single hKO1‐positive iPSCs were sorted using the FACSAria III system (BD Biosciences, San Jose, CA) and then expanded. 253G1‐hiPSCs expressing Tet‐inducible HSVtk (HSVtk‐hiPSCs) were dissociated into single cells, seeded in 96‐well plates at a density of 5 × 103 cells/200 μl per well with or without 1 μg/ml doxycycline (DOX; Wako Pure Chemical Industries, Ltd., Osaka, Japan). After 3 days of incubation, the cell viability assay was performed using the Cell Counting Kit‐8 (Dojindo Molecular Technologies, Kumamoto, Japan) as described previously 41 .
7
Apoptosis Induction in GBM Cells
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GBM cells seeded into the 6-well plates with different concentrations of VB (0, 20, 40, 60, 80, and 100 μΜ) and transfection were collected for this experiment according to the provided instructions of the Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) cell apoptosis detection kit (MA0220; Dalian Meilun Biotechnology, PR China). Briefly, cells were collected, rinsed with PBS, and counted. Then, the cells (2−5 × 105 cells/mL) were collected and centrifuged at 500 × g for 5 min. The supernatant was then discarded, and the cells were resuspended with 195 μL of the binding buffer. Thereafter, 5 μL Annexin V-FITC was added to the cells and incubated in conditions devoid of light for 10 min at room temperature, followed by addition of 10 μL PI (20 μg/mL) and incubation in conditions devoid of light for 5 min at room temperature. All cells were detected by a flow cytometer. The blank control group, PI single-staining group, and Annexin V-FITC single-staining group were set in the experiment. The apoptosis of cells was finally measured using the FACSAria III system (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was repeated three times.
8
Irradiated Mouse LSK Cell Profiling
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LSK cells were separated from mice aged 6–8 weeks 1 h after 4 Gy irradiation (n = 3/group) and sorted by flow cytometry using a FACSAria III system (BD Biosciences). Subsequent transcriptome sequencing was commissioned by Beijing Genomics Institute (China).
9
Analyzing Cell Proliferation in RACK1-Depleted HEK293
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HEK293 cells expressing RACK1-targeting shRNA or RACK1-HaloTag constructs were analyzed under conditions of active growth by comparison with controls. Before the analysis, we supplemented the medium with 10 μM bromodeoxyuridine (BrdU) (from the APC BrdU flow kit, produced by BD Biosciences) for 6 h. After that, if the cells were transfected with RACK1-HaloTag, they were incubated with the HaloTag TMR ligand (Promega) for 30 min at 37°C according to the manufacturer's instructions. Then we changed the medium to eliminate any excess of HaloTag TMR dye. We trypsinized and centrifuged the cells for 5 min at 500 × g. After that, we utilized the reagents and protocol from the APC BrdU flow kit to resuspend, fix, and permeabilize cells, to degrade the DNA in order to expose BrdU epitopes, to mark BrdU by staining with an allophycocyanin (APC)-conjugated antibody, and to stain total DNA with 7-aminoactinomycin D (7-AAD).
We used a FACSAria III system (BD Biosciences) to measure fluorescence in cells and FlowJo, v10.3 (Tree Star), to perform analysis.
We used a FACSAria III system (BD Biosciences) to measure fluorescence in cells and FlowJo, v10.3 (Tree Star), to perform analysis.
10
Cell Death Assay with AZD3965
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On day 0, cells were seeded at 40,000 to 100,000 cells per well in a 24 multiwell plate for 16 h, in DMEM containing no glucose and no glutamine (Sigma-Aldrich catalogue # D5030), supplemented with 10 mmol/L sodium L-lactate (Sigma-Aldrich) and 1% FBS. Cells were then treated ±10 µmol/L of AZD3965 and cultured up to 7 days. On each day, cell death was determined using the Annexin V Apoptosis Detection kit FITC (ThermoFisher catalogue #88-8005-74) according to manufacturer’s recommendations. Flow cytometric profiles were determined on a FACS AriaIII system (BD Biosciences). A minimum of 5000 events were acquired for each sample. Analyses were performed using the FlowJo software (BD Biosciences), where doublets were removed and single cells then categorized according to their Annexin V and propidium iodide status.
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