Cell surface expression of β1 and β3 integrins was performed by staining adherent cells that had been seeded on 10 µg/ml FN for 3 h under tissue culture conditions. Cells were stained with Alexa Fluor 488–conjugated antibodies against β1 or β3 integrin (BioLegend) for 15 min in serum-containing medium in a tissue-culture incubator per the supplier’s recommendation. Cells were trypsinized, pelleted, filtered through a 30-µm filter (Sysmex), and placed on ice before immediately profiling populations in a Bio-Rad S3 flow cytometer. Cytoplasts were initially profiled for purity using the Vybrant DyeCycle Green stain to ensure purity. Median integrin–Alexa 488 fluorescence values for each integrin were divided by the median forward scatter values for each respective population to provide a relative integrin/particle size ratio. Differences in relative integrin levels were not detected between intact cells and cytoplasts when either mean or geometric mean values for integrin–Alexa 488 fluorescence/forward scatter were measured. Values were measured from two independent experiments containing technical duplicates.
S3 flow cytometer
Manufactured by Bio-Rad
Sourced in United States
The S3 flow cytometer is a compact, benchtop instrument designed for cell analysis and sorting. It utilizes advanced laser and optic technology to rapidly detect, identify, and sort individual cells within a sample. The S3 flow cytometer is capable of analyzing multiple parameters of each cell, such as size, granularity, and the presence of specific fluorescent markers.
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Lab products found in correlation
Prosort software, by Bio-Rad (2 mentions)
Anti mouse 488 alexa anti mouse 488, by Thermo Fisher Scientific (2 mentions)
Fcs express, by De Novo Software (2 mentions)
Prosort, by Bio-Rad (2 mentions)
Mouse anti brdu antibody, by BD (2 mentions)
Mitotracker red cm h2xros, by Thermo Fisher Scientific (1 mentions)
Fcs express software, by De Novo Software (1 mentions)
Anti brdu, by BD (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Annexin 5 fitc conjugate, by Thermo Fisher Scientific (1 mentions)
Ptagrfp c, by Evrogen (1 mentions)
10 protocols using s3 flow cytometer
1
Integrin Expression Profiling in Cells
2
Mitochondrial Membrane Potential Assay
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MO3.13 cells were seeded in 24 well plates (25000 cells/cm2) and treated as described before; after treatments, the cells were stained with MitoTracker Red CM-H2XRos (Molecular Probes-Life Technologies, Carlsbad, CA, USA) for 30 min at 37 °C in humidified atmosphere containing 5% CO2. MitoTracker Red CM-H2XRos was diluted with anhydrous DMSO to obtain 1 mM of stock solution and used 1 μM as final concentration. After staining, cells were harvested and spun for 5 min at 1400 rpm; the obtained pellets were washed with PBS and spun again for 5 min at 1400 rpm. Flow cytometry was performed by using a S3 flow cytometer (Bio-Rad) equipped with a 488 nm and 561 diode-pumped solid-state lasers. MitoTracker Red CM-H2XRos was excited with a 561 nm laser and fluorescence emission was collected through a 605/40 nm band-pass filter and 570 nm long-pass fiter. At least 20 000 gated events were acquired for each sample. Data were analyzed using the Bio-Rad ProSort Software.
3
Multicolor Flow Cytometry Analysis
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Cells were harvested after mild trypsin treatment and were washed and re-suspended in PBS and FBS 1% to inhibit trypsin activity. Flow cytometry was performed by using a BD LSRII flow cytometer ((BDBioscience, Becton Dickinson, USA)) equipped with 405 and 488 nm lasers. mBFP was excited with the 405 nm laser and its fluorescence was collected through a 440/40 nm band-pass filter band-pass filter. mGFP was excited using the 488 nm laser, and the emitted fluorescence was collected through a 530/30 nm band-pass filter. In case of the construct mCherry-APP-mGFP, flow cytometry was performed by using an S3 flow cytometer (Bio-Rad) equipped with 488 and 561 nm diode-pumped solid-state lasers. mGFP was excited using the 488 nm laser, and the emitted fluorescence was collected through a 525/30 nm band-pass filter; mCherry was excited with the 561 nm laser and its fluorescence was collected through a 615/25 nm band-pass filter.
Data were analyzed using the free Flowing software (Cell Imaging and Cytometry Core, Turku Bioscience Center, Finland). The cellular debris was excluded from the quantification by gating cells on the Forward scatter area/Side scatter area (FSC/SSC). Approximately 10000 events were acquired for each sample.
Data were analyzed using the free Flowing software (Cell Imaging and Cytometry Core, Turku Bioscience Center, Finland). The cellular debris was excluded from the quantification by gating cells on the Forward scatter area/Side scatter area (FSC/SSC). Approximately 10000 events were acquired for each sample.
4
BrdU Incorporation and Cell Cycle Analysis
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Cells were fixed in cold 70% ethanol at the indicated times after 30 μM bromodeoxyuridine (BrdU; Sigma-Aldrich) incubation for 30 min and stored at 4°C. DNA was denatured with 2N HCl and 0.5% Triton X-100 and then neutralized with 0.1 M sodium borate (pH 8.5). The pellet was resuspended with 0.5% Tween 20 and 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), anti-BrdU (Becton Dickinson, Franklin Lakes, NJ, USA) was added for 1 h, followed by 1 h of anti-mouse-488 (Alexa anti-mouse 488, Molecular Probes/Invitrogen). The samples were incubated with 10 μg/ml propidium iodide and analysed on a S3 flow cytometer (Biorad, Hercules, CA, USA). The plots were analysed using FCS Express software (De Novo Software). For single propidium iodide staining, the cells were fixed as above before resuspension in PBS-0.1% Tween-20, 50 μg/ml propidium iodide, 5 μg/ml RNase A. After incubation for 15 min, the samples were analysed by FACS. Cell cycle distribution was fitted to a single cycle model with FCS Express.
5
BrdU Incorporation and Cell Cycle Analysis
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Cells were harvested after incubation with 30 μM bromodeoxyuridine (BrdU; Sigma–Aldrich) for 30 min, fixed in 1 ml of cold 70% ethanol and stored at 4°C. DNA was denatured with 2N HCl and 0.5% Triton X-100, then neutralized with 1 ml of 0.1M sodium borate pH8.5. The cell pellet was resuspended in PBS, 0.5% Tween 20, 1% bovine serum albumin (BSA) with a mouse anti-BrdU antibody (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated at room temperature for 1 h, followed by 1-h room temperature incubation with anti-mouse-488 (Alexa anti-mouse 488, Molecular Probes/Invitrogen). Samples were finally incubated with 10 μg/ml Propidium Iodide and analyzed on a S3 flow cytometer (Biorad). Data were analyzed using the program ProSort (Biorad) or FCS Express (DeNovo). When Propidium Iodide only staining was performed, cells were fixed in 1 ml of cold ethanol and resuspended in PBS, 0.1% Tween 20, 50 μg/ml Propidium Iodide, 5 μg/ml RNase A. After incubation at 37°C for 15 min, cells were analyzed by flow cytometry.
6
BrdU Pulse-Chase Assay for DNA Replication
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The U2OS and BJ-hTERT cells were pulsed with 30 µM BrdU ((Sigma Aldrich) for 30 min or 1 h and the DNA replication was followed after its withdrawal, respectively, for 0–6–9 or 12 h in cells transfected with scrambled siRNA or after silencing of POLL. The cells were then fixed in 1 mL of cold 70% ethanol and stored at 4 °C overnight. DNA was denatured with 2N HCl/0.5% Triton X-100 and neutralized with 1 mL of 0.1M sodium borate pH8.5. After centrifugation at 500g for 10 min, the cell pellet was resuspended with a mouse anti-BrdU antibody (1:100; Becton Dickinson,), diluted in 1% BSA, PBS 0.5% Tween 20, and incubated for 1 h at room temperature. Cells were then centrifuged at 500 g for 10 min and the pellet was resuspended with anti-mouse-488 (1:500; Alexa anti mouse 488, Molecular Probes/Invitrogen) and incubated for 1 h at room temperature. Samples were finally incubated with 10 ug/mL Propidium Iodide and analysed on a S3 flow cytometer (Bio-rad). Data were analysed using the program ProSort (Bio-rad) or FCS Express (DeNovo).
7
Annexin V-FITC and PI Apoptosis Assay
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After treatments, MO3.13 cells were harvested and spun for 5 min at 1400 rpm; the obtained pellets were washed with PBS 1 × and spun again for 5 min at 1400 rpm. Cells were re-suspended in binding buffer (10 mmol/l HEPES, 135 mmol/l NaCl, 5 mmol/l CaCl2) containing Annexin V-FITC conjugate 1 μM (Invitrogen-Life Technologies, Carlsbad, CA, USA) and PI 100 nM (Sigma-Aldrich, St. Louis, MO, USA) and incubated in ice for 30 min. Flow cytometry was performed by using a S3 flow cytometer (Bio-Rad, Hercules, CA, USA) equipped with 488 and 561 nm diode-pumped solid-state lasers. Annexin V-FITC was excited using the 488 nm laser, and fluorescence was collected through a 552/50 nm band-pass filter and 505 nm long-pass filter; PI was excited with the 561 nm laser and the fluorescence was collected through a 605/40 nm band-pass filter and a 570 nm long-pass fiter. At least 20 000 gated events were acquired for each sample. Data were analysed using the Bio-Rad ProSort software.
8
Evaluation of DNA Content by Flow Cytometry
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Flow cytometry analyses to evaluate DNA content were performed as previously described [35 (link)]. Cells were fixed in 70% ethanol and then stained with 50 μg/ml propidium iodide in PBS-0.1% Tween supplemented with 5 μg/ml of RNaseA. Samples were then analyzed using an S3 flow cytometer (Biorad, Hercules, CA, USA) and FlowJo Software (BD Biosciences).
9
Flow Cytometric Analysis of Enucleation
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Cells were suspended in PBS (Ca2+/Mg2+ free) containing 0.5% FBS and 5 mM EDTA and stained with Vybrant DyeCycle Green stain at a final concentration of 5 µM for 15 min at 37°C. Samples were then filtered through a 30-µm filter (Sysmex; Partec CellTrics) and placed on ice. Stained populations were individually profiled in a Bio-Rad S3 flow cytometer. For population analyses, ∼50,000 cells were profiled per sample. In addition to nuclear dye detection, cytoplasts were also identified from intact cells based on distinct side-scatter profiles. Periodically, this was used to assess cytoplast population purity. FlowJo (v10.1r5) software was used for graphic visualization of population distributions and extraction of statistical values. All fluorescence threshold values were designated based on unlabeled and labeled cells. Values reporting percent enucleation efficiency are based on seven independent enucleation runs.
10
Efficient Homozygous Mutant Generation
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In a single round of mutagenesis, we identified heterozygous, but not homozygous, insertions. Homozygous mutants were generated from gene conversion by transfecting heterozygous clones with the hCas9 plasmid, the gRNA, and pTagRFP-C (Evrogen) as above. 48 hours post transfection, cells positive for RFP were isolated using a BioRad S3 flow cytometer. Clonal cell lines were generated and PCR screened as described above.
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