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Facs aria model

Manufactured by BD

The BD FACS Aria is a high-performance cell sorter designed for research applications. It is capable of multi-parameter analysis and sorting of cells or particles based on their physical and fluorescent characteristics. The core function of the FACS Aria is to provide users with a powerful tool for cell sorting and analysis.

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7 protocols using facs aria model

1

Isolating and Characterizing Murine BMSC

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To separate BMSC from the medullary cavity, female mice were sacrificed, and BMSC were washed from the femora and treated at 4 °C for 20 min with FITC, PE, and allophycocyanin conjugated antibodies, as well as peridinin chlorophyll protein conjugated to CD29, CD45, CD11b, and Sca1 (BioLegend). Acquisition and analysis were carried out using the FACS Aria model and FACS DIVE software version 6.1.3 (BD Biosciences). Primary BMSC were isolated and planted in culture flasks for cell population enrichment. They were subcultured about 1 week later, when the secondpassage BMSC clustered. The osteogenesis induction media (5 mmol/L glycerophosphate and 50 g/mL ascorbic acid, 300 ng/mL BMP2) was applied to BMSC for 48 h in 24-well plates (5 × 105 cells/well). Then, homogenize the cell lysates to determine ALP activity using the enzymatic colorimetric ALP Kit (from Roche) and spectrophotometric determination of the output of pnitrophenol. The amounts of secreted osteocalcin were determined in culture media using an immunoassay kit (DiaSorin).
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2

Isolation and Differentiation of Mouse and Human BMSC

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We isolated and collected BMSC of mouse as before, as well as the cultivation.18 In order to isolate BMSC from medullary cavity, female mice were killed, and BMSC were washed out from femora and incubated at 4°C for 20 minutes with FITC‐, PE‐ and allophycocyanin‐conjugated antibodies, and peridinin chlorophyll‐protein that combined with CD29, CD45, CD11b and Sca‐1 (BioLegend). For the isolation of human BMSC, we use the same method to harvest human BMSC. The human BMSC were incubated at 4°C for 30 minutes with antibody of allophycocyanin‐, FITC‐ and PE‐conjugated which recognized CD45, CD146 and STRO‐1 (BioLegend). By using FACS Aria model and FACS DIVE software version 6.1.3(BD Biosciences), acquisition was carried out and the analysis was enforced.
Here, we find out that the mouse BMSC were sorted as CD29+Sca‐1+CD45−CD11b‐, while human BMSC (hBMSC) were sorted as CD146+STRO‐1+CD45‐. Then, we gathered and cultured them for 1‐2 weeks. In culture flasks, the primary BMSC were separated and seeded for cell population enrichment. Approximately 1 week later, as the second‐passage BMSC reached clustered, they were subcultured. Afterwards, we induced adipogenic and osteogenic differentiation in the third‐passage BMSC. Plasmid transfection also executed in third‐passage BMSC.
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3

Isolation and Purification of Intestinal Stem Cells

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Crypts were isolated using an established protocol4 (link)5 (link)6 (link)7 (link)41 (link). First, intestines were flushed with phosphate-buffered saline (PBS) and incised longitudinally after which villi were removed mechanically by scraping. Sections (1 cm) were incubated in EDTA (5 mM)/PBS for 15 min at 4 °C per fraction of epithelium. After incubation, the epithelium was separated by vigorous shaking and the remaining intestinal tissue was placed in a new tube for collection of subsequent fractions. After isolation, crypt cells were pelleted, passed through a 70 μm cell strainer (unless indicated otherwise), evaluated for purity microscopically and counted. For purification of single Lgr5-GFP+ ISCs, isolated crypts were incubated in culture medium for 45 min at 37 °C, followed by trituration with a glass pipette. Dissociated cells were passed through a cell strainer with a pore size of 20 μm. GFP+ and GFP− cells were sorted by FACS Aria model (BD Biosciences).
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4

Isolation and Characterization of Murine Mesenchymal Stem Cells

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After euthanization, we collected bone marrow cells from 6-week-old male wild-type mice and cultured them in alpha minimum essential medium (α-MEM, Mediatech, Inc.) containing 100 U ml−1 penicillin (Sigma-Aldrich), 100 μg ml−1 streptomycin sulfate (Sigma-Aldrich) and 20% lot-selected fetal bovine serum (FBS, Atlanta Biologicals) at 37 °C in a 5% CO2 humidified incubator. After 72 h, we removed non-adherent cells and cultured adherent cells for an additional 7 d with a single media change. Then, we incubated cell aliquots for 20 min at 4 °C with fluorescein isothiocyanate-, phycoerythrin-, peridinin chlorophyll protein- and allophycocyanin-conjugated antibodies to mouse CD29 (Biolegend, HMβ1-1), Sca-1 (Biolegend, D7), CD45 (Biolegend, 30-F11) and CD11b (Biolegend, M1/70). We performed acquisition on a FACS Aria model (BD Biosciences), and did the analysis using FACS DIVE software version 6.1.3 (BD Biosciences). We sorted CD29+Sca-1+CD45CD11b cells and enriched them by further culture.
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5

Isolation and Culture of Mouse BMSCs

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We collected the bone marrow cells from the tibiae of rGDF11- or vehicle-treated animals by flushing. Cells were incubated in the red blood cell lysis buffer for 5 min at room temperature. The sorting of BMSCs were performed as described61 (link). Briefly, the cell aliquots were incubated with phycoerythrin (PE)-, fluorescein isothiocyanate (FITC)-, peridinin chlorophyll protein (Per CP)-, and allophycocyanin (APC)-conjugated antibodies against mouse CD29, Sca-1, CD11b and CD45 (BioLegend, San Diego, CA) at 4 °C for 30 min. Cell sorting was performed on a fluorescence-activated cell sorting (FACS) Aria model (BD Biosciences), and analysis was performed with fluorescence-activated cell sorting DIVE software version 6.1.3 (BD Biosciences).
The sorted Sca-1+CD29+CD45CD11b cells were collected and plated into 6-well plates (1,000 cells per well). After 4 h of adhesion, unattached cells were removed. Medium was changed every 3 days, and cultures were fixed and stained with 0.5 mg ml−1 of crystal violet on day 10. Colonies containing 50 or more cells were counted.
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6

Isolation and Characterization of Murine Mesenchymal Stem Cells

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After euthanization, we collected bone marrow cells from 6-week-old male wild-type mice and cultured them in alpha minimum essential medium (α-MEM, Mediatech, Inc.) containing 100 U ml−1 penicillin (Sigma-Aldrich), 100 μg ml−1 streptomycin sulfate (Sigma-Aldrich) and 20% lot-selected fetal bovine serum (FBS, Atlanta Biologicals) at 37 °C in a 5% CO2 humidified incubator. After 72 h, we removed non-adherent cells and cultured adherent cells for an additional 7 d with a single media change. Then, we incubated cell aliquots for 20 min at 4 °C with fluorescein isothiocyanate-, phycoerythrin-, peridinin chlorophyll protein- and allophycocyanin-conjugated antibodies to mouse CD29 (Biolegend, HMβ1-1), Sca-1 (Biolegend, D7), CD45 (Biolegend, 30-F11) and CD11b (Biolegend, M1/70). We performed acquisition on a FACS Aria model (BD Biosciences), and did the analysis using FACS DIVE software version 6.1.3 (BD Biosciences). We sorted CD29+Sca-1+CD45CD11b cells and enriched them by further culture.
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7

Isolation of Mouse and Human SSCs

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For mouse SSC isolation, bone marrow from WT and knockout mice was flushed out using a 25 gauge needle. Briefly, cells were digested with trypsin for 2min at 37˚C, and passed through a 40 μm strainer. Cell were cultured in alpha modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) supplemented with 200 μM L-glutamine and non-essential amino acids for 2 weeks. Colony forming cells were harvested and washed twice with FACS buffer (PBS, 10mM EDTA, 2% FBS), resuspended and stained with anti-CD11b-FITC (1:100 BioLegend Cat#101205) anti-CD29-PE (1:100, Biolegend Cat#102207), anti-CD45-PerCP (1:100, Biolegend Cat#103129) and anti-Sca1-APC (1:100, BioLegend Cat#108111) for 30min on ice. Sca1+CD29+CD45CD11b SSCs were sorted by fluorescence-activated cell sorting (FACS) as described previously (Chang et al., 2013 (link)). Cell gating was based on comparison with isotype controls and single stained controls.
For human bone marrow SSC isolation, human bone marrow cells were incubated with anti-STRO-1-FITC (1:100, BioLegend Cat#340106), anti-CD45-APC (1:100, BioLegend Cat#304012;), and anti-CD146-PE (1:100, BioLegend Cat#361008) at 4˚C for 30 minutes. Acquisition of CD146+STRO-1+CD45 cells was performed on a FACS Aria model (BD Biosciences).
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