The 266-6 cells were cultured in 6-well plates for 18 hours to study the effect of OOS in the cell cycle in vitro. Then, cells were treated with 1:100, 1:200, and 1:500 (V/Vf) dilutions of OOS in complete RPMI for 48 hours. The control cells were cultured with the control medium. Then, cells were trypsinized, washed once with phosphate-buffered saline (PBS), and fixed with 70% ethanol for 15 minutes at 4°C. Afterwards, cells were washed with PBS 3 times and incubated with propidium iodide (PI) containing FxCycle PI/RNase Solution (Thermo Fisher Scientific) following the manufacturer's indications. Finally, cell cycle changes were analyzed by flow cytometry using the Gallios cytometer (Beckman Coulter, Brea, Calif). For cell division analysis, 266-6 cells were fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE) by incubating at 37°C for 30 minutes before seeding them into 6-well plates. Then, cells were treated as described above for cell cycle analysis. After 48 hours, cells were trypsinized, washed in PBS, and resuspended for flow cytometry studies by Gallios cytometer (Beckman Coulter).
Gallios cytometer
Manufactured by Beckman Coulter
Sourced in United States, France, Italy, Germany
The Gallios cytometer is a flow cytometry instrument designed for advanced multiparameter analysis of cells and particles. It features high-resolution optics, sensitive detectors, and a modular configuration to support a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Kaluza software, by Beckman Coulter (23 mentions)
Anti cd8α, by BD (5 mentions)
Anti tcrγδ, by BD (5 mentions)
Anti tcrαβ, by BD (5 mentions)
Anti cd4, by BD (5 mentions)
Ionomycin, by Merck Group (4 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (4 mentions)
Mitotracker green, by Thermo Fisher Scientific (4 mentions)
Anti cd45ra, by BD (4 mentions)
Anti cd8β, by BD (4 mentions)
Anti nkr p1a, by BD (4 mentions)
Fxcycle pi rnase solution, by Thermo Fisher Scientific (3 mentions)
Kaluza analysis software version 2, by Beckman Coulter (3 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Cytomics fc500, by Beckman Coulter (3 mentions)
Anti cd103, by BioLegend (3 mentions)
Anti cd62l, by BioLegend (3 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Fixable viability dye efluor 450, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm, by BD (2 mentions)
134 protocols using gallios cytometer
1
OOS Effect on Cell Cycle and Division
2
Multivalent Fluorescent Cell Labeling
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To evaluate the acidic pH-powered in situ assembly multivalent system for fluorescent labeling of target cells, equal DM1I, DM2I and DM3I (15 nM) were co-incubated with 1.5 × 105 target tumor cells in binding buffer (pH 7.4 or 6.3) at 37 °C for 1 h. Subsequently, it was determined by a Gallios cytometer (Beckman Coulter, USA) by counting 10,000 events.
To evaluating the binding capacity of TA-aptNC-based in situ assembly strategy at cellular level, different probes were reasonably designed, including TA-CaptNC (the mixture of CDM1I, CDM2I and CDM3I, aptamer sequence replaced by arbitrary sequences), pH-C (the mixture of DM1C, DM2C and DM3C, split i-motif sequence substituted by pH-insensitive sequence), pH-aptDM (the corresponding monovalent probe) and TA-aptNC (the mixture of DM1I, DM2I and DM3I). These probes were respectively treated with the target cells and then determined by a Gallios cytometer (Beckman Coulter, USA).
To evaluating the binding capacity of TA-aptNC-based in situ assembly strategy at cellular level, different probes were reasonably designed, including TA-CaptNC (the mixture of CDM1I, CDM2I and CDM3I, aptamer sequence replaced by arbitrary sequences), pH-C (the mixture of DM1C, DM2C and DM3C, split i-motif sequence substituted by pH-insensitive sequence), pH-aptDM (the corresponding monovalent probe) and TA-aptNC (the mixture of DM1I, DM2I and DM3I). These probes were respectively treated with the target cells and then determined by a Gallios cytometer (Beckman Coulter, USA).
3
Flow Cytometric Analysis of B-Cell Phenotype
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Cells were stained to verify the purity of isolated B cells or to define B-cell phenotype by using anti-CD38, anti-CD24, anti-CD27, anti-CD71, anti-CD80, anti-CD86, anti-CD20 (Beckman Coulter, Brea, CA, USA), PD-1 (Miltenyi Biotech), PD-L1 and anti-TIM-1 (eBiosciences) antibodies. For intracellular labelling of IL-10 (Miltenyi Biotech), the B-cell cultures were supplemented with Phorbol 12-myristate 13-acetate (PMA; 10 ng/mL) + Ionomycin (0.25 µg/mL, both from Sigma Aldrich) during 5 h and GolgiStop (BDbiosciences; Franklin Lakes, NJ, USA) during the last 4 h of incubation. Then, cells were washed, surface-stained, stained for viable cells with Fixable Viability Dye eFluor450 (eBiosciences), fixed/permeabilised (Cytofix/Cytoperm, BDbiosciences) and stained for intracellular IL-10. Cells were then analysed by flow cytometry using a Gallios cytometer (Beckman Coulter), and data were analysed using the Kaluza software (Beckman Coulter).
4
Multiparameter Flow Cytometry Analysis
5
Multiparametric Immune Cell Profiling by Flow Cytometry
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MLNLs (500,000 cells/tube) were stained using mouse anti-rat monoclonal antibodies conjugated to FITC, phycoerythrin (PE), peridinin-chlorophyll-protein (PerCP), allophycocyanin (APC) or APC-cyanine (Cy)7. The antibodies used herein were anti-TCRαβ, anti-CD8α, anti-CD4, anti-TCRγδ and anti-CD45RA (BD Biosciences, San Diego, CA, USA). Cells were mixed with PBS containing 2% FBS and 1% NaN3 and stained, as previously described [28 (link)]. The data were acquired with a Gallios™ Cytometer (Beckman Coulter, Miami, FL, USA) in the CCiT-UB and assessed by the Flowjo v10 software (Tree Star, Inc., Ashland, OR, USA). Results are expressed as percentages of positive cells in the lymphocyte population, selected according to their forward-scatter characteristics (FSC) and side-scatter characteristics (SSC).
6
Evaluating Chemoresistance in Pancreatic Cancer
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Human pancreatic cancer cells were cultured in a complete medium for 24 hours in 6-well plates at 3 × 105 cells/well concentration. Once the cells were attached, the old medium was replaced with fresh medium for control condition or with 1:2 diluted fibroblast CM for the chemoresistance studies and incubated for 24 hours. Afterward, cells were treated with OOS 1:50 (V/Vf), combined with 1 μM paclitaxel and 1 μM gemcitabine diluted in fresh medium or fibroblast CM for 48 hours. Then, cells were washed, trypsinized, pelleted by centrifugation, washed again with PBS, and double stained with the annexin V–fluorescein isothiocyanate apoptosis detection kit and PI following manufacturer's instructions (both purchased from Thermo Fisher Scientific). Finally, apoptosis was determined by the Gallios cytometer (Beckman Coulter). Note that the HPAF-II cell line was not included in this assay because of the high levels of cell death during the process.
7
Flow Cytometric Analysis of MenSCs
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MenSCs (passage 3) were analyzed by flow cytometry for expression of the different cell surface markers. Fluorescein isothiocyanate- (FITC-) conjugated antibodies against CD29 (Immunostep, Salamanca, Spain), CD90 (eBioscience, San Diego, CA, USA), HLA-DR, CD14, CD34, CD83, CD86 (BD Biosciences, San Jose, CA, USA), CD40 (Biolegend, San Diego, CA, USA), phycoerythrin- (PE-) conjugated antibody against CD73 (BD biosciences), CD105a (eBiosciences) CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany), and Alexa Fluor 700-conjugated antibody against CD44 (BD Biosciences) were used. The corresponding fluorescent, isotype-matched negative control antibodies defined background staining.
Harvested MenSCs were washed with PBS containing 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) and 0.1% sodium azide (Sigma-Aldrich). Aliquots of 2 × 105 cells were next incubated on ice with the corresponding antibody, at the concentration recommended by the manufacturer, for 20 min and under light protection. A Gallios cytometer (Beckman Coulter, Brea, CA, USA) was used, and 10,000 events were analyzed for each sample. FlowJo v7.6.5 (Tree Star Inc., San Carlos, CA, USA) software was used for the analysis of the data.
Harvested MenSCs were washed with PBS containing 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) and 0.1% sodium azide (Sigma-Aldrich). Aliquots of 2 × 105 cells were next incubated on ice with the corresponding antibody, at the concentration recommended by the manufacturer, for 20 min and under light protection. A Gallios cytometer (Beckman Coulter, Brea, CA, USA) was used, and 10,000 events were analyzed for each sample. FlowJo v7.6.5 (Tree Star Inc., San Carlos, CA, USA) software was used for the analysis of the data.
8
Comprehensive Flow Cytometry Analysis of Regulatory T Cells
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The percentage of Treg and Tr1 cells was evaluated by flow cytometry on whole PB samples. Treg were identified using Human Regulatory T Cell Cocktail (BD Biosciences, Milan, Italy), containing anti-CD4 FITC, anti-CD25 PE-Cy7, and anti-CD127Alexa Fluor 647 antibodies, following manufacturer's protocol. Tr1 were evaluated using anti-CD4 PE-Cy7 (eBiosciences, San Diego, CA), anti-CD45R0 APC (eBiosciences), anti-CD49b PE (R&D Systems, Space, Milan, Italy), and anti-LAG-3 FITC (R&D Systems) antibodies. Samples were subjected to erythrocytes lysis using BD FACS lysis (BD Biosciences) and washed. Cells were run on Gallios cytometer (Beckman Coulter, Cassina De' Pecchi, MI, Italy) acquiring at least 104 events. Data were analyzed using Kaluza software (Beckman Coulter). Treg were identified as CD4+CD25highCD127low cells, whereas Tr1 were identified as CD4+CD45R0+CD49b+LAG-3+ cells [25 (link)].
9
Characterization of Murine Lymphocyte Subsets
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Lymphocytes from MLNs were extracellularly and intracellularly stained as previously reported [16 (link)]. Mouse anti-rat monoclonal antibodies (mAb) conjugated to fluorochromes—fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), allophycocyanin (APC) or brilliant violet 421 (BV421): FITC-TCRαβ, FITC-CD8β, FITC-CD25, PE-CD161a, PE-TCRγδ, PE-CD4, PerCP-CD8α, APC-CD4, and BV421-CD45RA (BD Biosciences) and APC-FoxP3 (eBioscience, Frankfurt, Germany). For extracellular staining, cells were incubated with saturating amounts of mAb in PBS containing 2% Fetal Bovine Serum (FBS) and 0.1% NaN3. For intracellular staining, cells were previously extracellularly labeled with anti-CD4-PE and anti-CD25-FITC mAb, then treated with Foxp3 fixation/permeabilization kit (eBioscience) and finally intracellularly stained with anti-Foxp3-APC mAb. Cells were fixed with 0.5% p-formaldehyde and stored at 4 °C in darkness until analysis by FCM. A negative control staining without any mAb and a staining control for each mAb were included.
Analyses were performed using a Gallios Cytometer (Beckman Coulter, Miami, FL, USA) in the FCU of CCiT-UB and by Flowjo v10 software.
Analyses were performed using a Gallios Cytometer (Beckman Coulter, Miami, FL, USA) in the FCU of CCiT-UB and by Flowjo v10 software.
10
Multiparameter Flow Cytometry Protocol
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Multi‐dimensional flow cytometric acquisition was performed using a Gallios cytometer (Beckman Coulter, Miami, FL). An effort was made to acquire a total of 1 million events per sample at rates not exceeding 3000 events/second. For DAPI staining, photomultiplier tubes were gain optimized for linear (cell cycle) detection of 2N cells 12 , 13 , 14 . The cytometer was calibrated to predetermined photomultiplier target channels prior to each use using SpectrAlign beads (DAKO, Cat. No. KO111) and 8‐peak Rainbow Calibration Particles (Spherotech, Libertyville, IL, Cat. No. RCP‐30‐5A). Offline compensation and analyses were performed using VenturiOne software designed for multidimensional rare event problems (Applied Cytometry, Dinnington, Sheffield, U.K.). Spectral compensation matrices were calculated for each experiment using single‐stained mouse IgG capture beads (Becton Dickinson, Cat. No. 552843) for each tandem antibody and hard stained beads (Calibrite, BD) for single molecule dyes (Becton Dickinson, FITC, PE [Cat.No. 349502], APC [Cat.No. 340487]).
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