The S-phase analysis was carried out by flow using EdU staining assay according to the manufacturer’s instructions. In brief, 2 × 105 M059K cells per well were seeded into 6-well plates and allowed to attach overnight. Cells were then treated with sc-13 or NU-7441, fixed with 4% formaldehyde in PBS, washed with 1% BSA and incubated for 30 min with Click-iT EdU reaction solution. After incubation, samples were washed, resuspended in 1% BSA, and analyzed with a BD FACS Calibur device and analyzed with FCS express V3 (BD Biosciences, USA).
Fcs express v3
Manufactured by BD
Sourced in United States
FCS Express V3 is a software application designed for the analysis and visualization of flow cytometry data. It provides tools for data processing, gating, and generating plots and reports.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur device, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Facscanto 2, by BD (1 mentions)
Annexin 5 fitc pi staining kit, by Abcam (1 mentions)
Rnaase, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
Golgiplug, by BD (1 mentions)
5 protocols using fcs express v3
1
Quantifying S-phase Cell Dynamics
2
Analyzing Cell-Cycle Progression by EdU Staining
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The S-phase analysis was carried out by flow cytometry (BD FACSCanto™ II, BD Bioscience, USA) using EdU staining assay according to the manufacturer’s instructions. In brief, 2 × 105 M059K or M059J cells per well were seeded into six-well plates and allowed to attach overnight. Cells were then transfected with anti-miR-1193 or anti-miR-NC, fixed with 4% formaldehyde in PBS, washed with 1% BSA and incubated for 30 min with Click-iT EdU reaction solution. After incubation, samples were washed, resuspended in 1% BSA, and analyzed with a BD FACSCalibur device and analyzed with FCS express V3 (BD Biosciences, USA).
3
Flow Cytometric Apoptosis Assay with Annexin-V/PI
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The flow cytometric apoptosis assay was carried out with an Annexin-V FITC/PI staining kit (ab14085, Abcam, UK) according to the manufacturer’s instructions. In brief, 2 × 105 M059K or M059J cells per well were seeded into six-well plates and allowed to attach overnight. Cells were then transfected with anti-miR-1193 or anti-miR-NC and incubated for 96 h in a humidified CO2 incubator. Subsequently, cells were trypsinized and centrifuged at 1000 rpm for 5 min. The obtained cell pellet was washed with DPBS and stained with both Annexin-V FITC and PI for 15 min. The stained cells were washed again with DPBS to remove excess dye and were finally resuspended in 300 μl of 1× PBS. Apoptotic events were analyzed, and untreated cells were used as the negative control for gating. A total of 10,000 events were recorded during the experiment. The percentages of live, early apoptotic, late apoptotic, and necrotic cells were analyzed with a BD FACSCalibur device and analyzed with FCS express V3 (BD Biosciences, USA).
4
ADMSCs Cell Cycle Analysis
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The ADMSCs were seeded at 0.2×106 cells/25 cm2 flask. At 80–90% confluence, the cells were harvested for cell cycle analysis. Briefly, the cells were washed and fixed in 70% ethanol overnight at −20°C. The fixed cells were then washed and incubated in 100 µg/ml propidium iodide (PI) and 20 ng/ml RNAase (both from Sigma-Aldrich) in phosphate-buffered saline (PBS) for 30 min. Cell cycles were assessed by flow cytometry, and analysis was performed using FCS Express V3 software (BD Biosciences, San Jose, CA, USA).
5
Quantifying IL-17 Producing PBMC Subsets
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The standard technique of Ficoll–Hypaque density centrifugation of heparinized blood was used to obtain peripheral blood mononuclear cells (PBMCs) from the study subjects. PBMCs (4 × 106) were cultured in RPMI 1640 supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mM β-mercaptoethanol, and 5% fetal bovine serum (FBS, GE Healthcare HyClone™, Utah, USA) in 24-well plates and stimulated with 50 ng/mL phorbol 12-myristate 13-acetate and 750 ng/mL ionomycin (Sigma–Aldrich, St. Louis, MO, USA) [17 (link)] for one hour before the addition of 1% GolgiPlug (BD Sciences, San Jose, CA, USA). After 4 hours of culture, PBMCs were harvested and stained with FITC anti-human CD4 or CD8, fixed and permeabilized with a fixation/permeabilization reagent, and finally stained with Phycoerythrin (PE) anti-human IL-17A (eBioscience, San Diego, CA, USA). Samples were run on BD FACSCalibur, and FCS ExpressV3 Software (BD Biosciences) was used for the analysis.
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