PLX cells were thawed, washed, resuspended in FACS buffer (phosphate buffered saline (PBS); Biochrom, Berlin, Germany) with 1% FBS), and aliquoted for immunostaining (∼200,000 cells per tube). Manufacturer recommended concentrations of fluorescein (FITC)‐ or phycoerythrin (PE)‐conjugated mouse anti‐human monoclonal antibodies (mAbs) (Becton Dickinson, Heidelberg, Germany) of interest (mesenchymal markers endothelial and leukocyte markers or their isotypes) were added, and cells were incubated at room temperature for 15 minutes. Cells were washed twice before they were subjected to flow cytometry in a Beckman Coulter FC‐500 system. Representative flow cytometry charts of PLX are illustrated in Supporting Information Figure 1.
Fc500 system
Manufactured by Beckman Coulter
Sourced in United States
The FC500 system is a flow cytometry instrument designed for cell analysis and sorting. It utilizes laser-based technology to detect and characterize various properties of cells or particles in a sample. The system is capable of measuring parameters such as size, granularity, and fluorescence intensity.
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Lab products found in correlation
Carboxyfluorescein diacetate succinimidyl ester cfse, by Thermo Fisher Scientific (1 mentions)
Anti cd11c pe mab, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ apc mab, by Thermo Fisher Scientific (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Cd133, by Miltenyi Biotec (1 mentions)
7 aad, by Beckman Coulter (1 mentions)
9 protocols using fc500 system
1
Phenotyping of Mesenchymal Stromal Cells
2
FACS Analysis of CD34+ Cell Counts
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Fluorescence-activated cell sorting (FACS) analysis was done with a FC500 system (Beckman Coulter, Brea, CA, USA), and the number of injected CD34+ cells in the BMMNC suspension calculated based on the detected CD34+ concentration and the volume of injected cell suspension.
All data were expressed as mean ± SD. Wilcoxon pair test, Mann–Whitney test, and Spearman correlation coefficient were used for statistical analysis.
All data were expressed as mean ± SD. Wilcoxon pair test, Mann–Whitney test, and Spearman correlation coefficient were used for statistical analysis.
3
Dual Detection of Bcl-2 Expression and Phosphorylation
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The FlowCellect Bcl-2 Activation dual detection kit (Merck Millipore, Burlington, MA, USA) was used to determine Bcl-2 expression and -phosphorylation at serine 70. MCF-7- and A549 cells were washed with the supplied wash buffer and resuspended in fixation buffer for 20 min. Fixed cells were washed in 1× assay buffer. Ice-cold permeabilization buffer (0.5 mL) was added to the pellet for 10 min. Assay buffer (100 µL) containing anti-Bcl-2 antibody conjugated to AlexaFluor® 488 and an anti-pBcl-2 (ser70) conjugated to phycoerythrin were added to the cells for 60 min at 4 °C (protected from light). Cells were washed twice with assay buffer. Fluorescence at FL1 (Bcl-2 antibody) and FL3 (pBcl-2, Ser 70) were measured with a FC500 System flow cytometer (Beckman Coulter).
4
Multiparametric Immune Cell Analysis
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Hepatic and intestinal mononuclear cells or splenic cells were isolated and labeled with anti-CD11c-PE mAb, anti-NK1.1-PE mAb, anti-CD44-FITC mAb, anti-T cell receptor (TCR)-β-FITC mAb, anti-TCR-β-Allophycocyanin (APC) mAb, anti-TCR-γδ-APC mAb, anti-IFN-γ-APC-mAb (all from eBioscience, San Diego, California, USA), anti-TNF-α-APC-mAb (Biolegend San Diego, California, USA). Stained cells were assessed using a FC500 System (Beckman Coulter, Brea, California, USA) equipped with Summit 5.1 software. For the cytotoxic assay, target cells were labeled in advance with carboxyfluorescein diacetate succinimidylester (CFSE; Molecular Probes, Eugene, Oregon, USA) as indicated and analyzed using flow cytometry.
5
BrdU Incorporation Analysis in HAP1 Cells
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HAP1 cells were pre-incubated in 30 µM BrdU (Sigma-Aldrich) for 30 min and harvested by trypsinization and washed twice with cold phosphate-buffered saline (PBS). About 1 × 106 cells were fixed with cold 70% ethanol overnight. After centrifugation at 2000 × g for 5 min, cells were incubated in 0.5 ml 2 N HCl/0.5% Triton X-100 for 30 min, followed by a centrifugation for the pellet. After re-suspension with 0.1 M sodium tetraborate for 2 min, cells were incubated with Alexa488-conjugated antibody against BrdU (Invitrogen) for 1 h at RT. Next, the samples were washed once with PBS, followed by incubation with 10 µg/ml PI and 50 µg/ml RNase in 500 µl PBS for 30 min at room temperature. Samples were then analyzed by flow cytometry FC-500 system (Beckman Coulter). A data set collected for 20,000 cells.
6
Enumeration of Circulating Endothelial Progenitor Cells
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Immunophenotype (CD45 and CD34, Beckman Coulter, https://www.beckmancoulter.com ; or Miltenyi Biotec, https://www.miltenyibiotec.com ; CD133, Miltenyi Biotec; CD184/CXCR4: BD Biosciences) and cell viability (7-AAD, Beckman Coulter or PI, Miltenyi Biotec) were determined by flow cytometry (FC 500 System, Beckman Coulter or MACSQuant Analyzer, Miltenyi Biotec). The tests were run according to EP [14 ,15 ].
Endothelial progenitor cells (EPC) were determined using a commercial kit (“EPC enrichment and enumeration kit”, Miltenyi Biotec) specifically designed for the enumeration of circulating EPC from peripheral blood, cord blood, leukapheresis products, or EPC from BM, based on the expression of CD34, CD133 and CD309 (VEGFR-2/KDR).
Endothelial progenitor cells (EPC) were determined using a commercial kit (“EPC enrichment and enumeration kit”, Miltenyi Biotec) specifically designed for the enumeration of circulating EPC from peripheral blood, cord blood, leukapheresis products, or EPC from BM, based on the expression of CD34, CD133 and CD309 (VEGFR-2/KDR).
7
Assessing Cellular Oxidative Stress
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Termination of the experiment followed 6-h, 24-h, and 48-h after radiation. MCF-7- and A549 cells were resuspended in 1 mL PBS. Hydroethidine stock solution (1 µL) was added to cells and incubated for 15 min at 37 °C. Cells were analyzed using a FC500 System flow cytometer (Beckman Coulter).
8
Neutrophil Survival Assay with SkQ1
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Purified neutrophils (1 × 106/mL) were treated with SkQ1 and/or with mitochondrial debris and incubated in a final volume of 0.5 mL for 22 hours at 37°C in a 5% CO2 humidified incubator. Neutrophil survival was assessed after 22 h using annexin V staining. Briefly, cells were washed twice with PBS, incubated for 20 min in the dark at 37°C with annexin-V-fluorescein isothiocyanate and propidium iodide, and analyzed with a Beckman Coulter FC500 system as described in [21 (link)]. Neutrophil apoptosis is expressed as the percentage of annexin-V-positive and propidium-iodide-negative cells.
9
Macrophage Phagocytosis and Inflammatory Response to Nanoparticles
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RAW246.7 macrophages (5×104) were seeded in 24-well plates and cultured in DMEM with 10% FBS and incubated in an atmosphere of 5% CO2 overnight at 37°C. FITC-labeled PDA NPs (100 μg/mL) and PDA@SCM NPs (100 μg/mL) were added to the culture medium, and cultured for an additional 12 h. Then, macrophages were washed with PBS three times for complete removal of the free NPs in the medium. Macrophages were digested and centrifuged, and flow cytometry using an FC500 system (Beckman Coulter, Fullerton, CA, USA) was used to detect NP phagocytosis by macrophages.
To determine the release of inflammatory factors from macrophages induced by NPs, PBS was selected as a negative control. After inoculation of macrophages in a 24-well plate for 24 h, LPS (1 μg/mL), PDA NPs (100 μg/mL), or PDA@SCM NPs (100 μg/mL) were added to the medium and the culture continued for an additional 12 h. Supernatants were collected carefully. The TNF-α content in supernatants was measured using the ELISA kit.
To determine the release of inflammatory factors from macrophages induced by NPs, PBS was selected as a negative control. After inoculation of macrophages in a 24-well plate for 24 h, LPS (1 μg/mL), PDA NPs (100 μg/mL), or PDA@SCM NPs (100 μg/mL) were added to the medium and the culture continued for an additional 12 h. Supernatants were collected carefully. The TNF-α content in supernatants was measured using the ELISA kit.
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