ADSC surface marker analysis using flow cytometry was performed on a FACS Calibur unit (Becton–Dickinson Biosciences, San Jose, CA, USA). Cells were stained with phycoerythrin-conjugated antibodies for CD19, CD34, CD11b, CD105, CD73, CD90, CD45 and HLA-DR (Becton–Dickinson Biosciences). For osteogenic and adipogenic differentiation, ADSCs were incubated with MesenCult Osteogenic or Adipogenic Stimulatory Medium (STEMCELL Technologies, Vancouver, Canada) for 2–3 weeks. Osteogenic and adipogenic differentiation was evaluated using Alizarin Red S and Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) staining, respectively.
Facscalibur unit
Manufactured by BD
Sourced in United States
The FACSCalibur is a flow cytometry instrument manufactured by BD. It is designed for multiparameter analysis and sorting of a wide variety of cells and particles. The FACSCalibur can detect and measure multiple parameters, including cell size, granularity, and fluorescence, allowing for the identification and characterization of different cell populations.
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Lab products found in correlation
Annexin 5 fluorescein isothiocyanate fitc pi apoptosis detection, by BD (2 mentions)
Fitc mouse anti human cd24, by BD (2 mentions)
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Oil red o, by Merck Group (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
Epics profile 2, by Beckman Coulter (1 mentions)
Multicycle software, by Phoenix Pharmaceuticals (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Fitc conjugated anti cd14, by BD (1 mentions)
Fitc conjugated anti cd34 monoclonal antibody, by GeneTex (1 mentions)
Pe conjugated anti vegfr2 monoclonal antibody, by BD (1 mentions)
9 protocols using facscalibur unit
1
Analysis of ADSC Surface Markers
2
Cell Cycle Analysis by Flow Cytometry
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Cell cycle analysis of cultured cells was determined by harvesting at 72 h after treatment, fixation overnight with 70% ethanol at −20 °C, and staining with 20 mg/mL propidium iodide (PI) in a buffer (1% Triton X-100 and 100 mg/mL RNase A) for 30 min. The FACSCalibur unit (Becton Dickinson, Franklin Lakes, NJ, USA) with the ModFit LT version 2.0 software was used to determine the levels of DNA. All experiments were repeated at least three times.
3
Cell Cycle and Apoptosis Analysis
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Flow cytometry analysis was performed as previously described.22 (link) For cell cycle analysis, the cells were trypsinized and washed into single-cell suspensions and fixed with ice-cold 70% ethanol at −20°C overnight. The fixed cells were then subsequently stained with 20 mg/mL propidium iodide (PI) staining buffer (containing 1% Triton X-100 and 100 mg/mL RNase A) for 30 mins. DNA content was assessed using a FACSCalibur unit (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with ModFit LT v2.0 software. To analyze apoptosis, cultured cells were harvested by trypsinization and were washed with PBS. Cells (1×106) from each sample were processed for annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection according to the manufacturer’s instructions (Becton Dickinson, Franklin Lakes, NJ, USA). All the experiments were repeated at least three times.
4
Radiation-induced Cell Cycle and Apoptosis
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Cultured cells were harvested 24 h after receiving 6 Gy of IR and then cell cycle progression and apoptosis were analyzed by flow cytometry. For cell cycle analysis, the cells were trypsinized and washed in PBS for 5 min prior to collection by centrifugation at 1500 rpm. The cells were then fixed with ice-cold 70% ethanol at −20°C overnight. The fixed cells were subsequently stained with 20 mg/mL propidium iodide (PI) staining buffer (containing 1% Triton X-100 and 100 mg/mL RNase A) for 30 min. DNA content was assessed using a FACSCalibur unit (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with ModFit LT v2.0 software. For the apoptosis assay, the cells were harvested by trypsinization and were washed with PBS. The cells were resuspended in 1× binding buffer at a concentration of 3 × 106 cells/mL. After staining the cells with fluorescein isothiocyanate (FITC) Annexin V and PI, the cells were analyzed using an Epics Profile II flow cytometer (Beckman Coulter, Fullerton, CA, USA) and Multicycle software (Phoenix Flow Systems, San Diego, CA, USA). All of the experiments were repeated at least three times.
5
Cell Cycle and Apoptosis Analysis
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The cell cycle was analyzed by flow cytometry. Briefly, cultured cells were trypsinized into single-cell suspensions and fixed with 70% ethanol for 30 min on ice. RNA was degraded by incubation with 20 mg/mL RNase (Sigma–Aldrich, St. Louis, MO, USA) for 1 hour at 37°C. DNA was labeled with 20 mg/mL propidium iodide (PI, Sigma–Aldrich); DNA content was assessed using a FACSCalibur unit (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with ModFit LT v2.0 software. To analyze apoptosis, cultured cells were harvested by trypsinization and washed with PBS. Cells (1 × 106) from each sample were processed for annexin V–fluorescein isothiocyanate (FITC)/PI apoptosis detection according to the manufacturer's instructions (Becton Dickinson).
6
Isolation and Characterization of CD24+ Cells
7
Isolation and Characterization of CD24+ Cells
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Confluent NP cells were dissociated from 10 cm2 tissue culture dishes with accutase at 37°C and incubated with 10 U/ml DNase in Neurobasal medium for 10 minutes. The cells were passed through a 40 μm filter, centrifuged, and resuspended in 100 μL NPM with 5 μM EDTA and 0.5% bovine serum albumin (BSA) (about 1×106 cells). 20 μL (1 test) of mouse anti-human CD24-FITC (BD Biosciences, 560992) was added per sample and incubated in the dark on ice for 30 minutes. The cells were then washed twice and resuspended in NPM with EDTA and BSA at 2.5 ×105 cells/ml. Cells were stored on ice in the dark until analyzed on a FACSCalibur unit (BD Biosciences) omitting forward and side scatter. Cells incubated without antibody were used as a negative control to adjust settings above autofluorescence.
8
Circulating Endothelial Progenitor Cells Quantification
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To determine circulating EPC numbers, MNCs isolated from peripheral blood (PB) were stained with FITC-conjugated anti-CD34 monoclonal antibody (GeneTex, Inc., USA) and PE-conjugated anti-VEGFR2 monoclonal antibody (BD Biosciences). ‘Early EPCs’ were stained with FITC-conjugated anti-CD14 (BD Pharmingen) or AlexaFluor® 647- anti-CD45.2 antibody (BioLegend). A FACScalibur unit (BD Biosciences) was used to detect fluorescent labeled cells.
9
Cell Proliferation and Cell Cycle Analysis
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Cultured cells (1x10 5 /well) were plated in 96-well plates. Cell proliferation at 24, 48 and 72 h was evaluated using a BrdU Cell Proliferation Assay kit (cat. no. 11647229001; Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. The cell proliferation rate was determined by measuring the absorbance at 492 nm using a computer-controlled microplate reader (Multiskan FC; Thermo Scientific, Waltham, MA, USA) (13) .
Fluorescence-activated cell sorting analysis. Cells were collected, washed with 300 µl cold phosphate-buffered saline and precipitated with 3 ml cold 75% ethanol. Cells to be used for fluorescence-activated cell sorting (FACS) were stored at -20˚C in 75% ethanol for 16 h, stained with propidium iodide for 30 min at 37˚C and the DNA content analyzed using a FACS Calibur unit (BD Biosciences, Allschwil, Switzerland) (14) .
Fluorescence-activated cell sorting analysis. Cells were collected, washed with 300 µl cold phosphate-buffered saline and precipitated with 3 ml cold 75% ethanol. Cells to be used for fluorescence-activated cell sorting (FACS) were stored at -20˚C in 75% ethanol for 16 h, stained with propidium iodide for 30 min at 37˚C and the DNA content analyzed using a FACS Calibur unit (BD Biosciences, Allschwil, Switzerland) (14) .
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