Astrios sorter

Manufactured by Beckman Coulter

Sourced in United States

The Astrios sorter is a high-performance cell sorting instrument designed for advanced flow cytometry applications. It features a flexible configuration and advanced optics to provide efficient cell sorting capabilities.

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Lab products found in correlation

Rmgm csf, by BioLegend (3 mentions) Trypsin, by Thermo Fisher Scientific (3 mentions) Streptavidin magnetic beads, by Miltenyi Biotec (2 mentions) Biotinylated mouse ly6g abs, by BioLegend (2 mentions) Macs column purification, by Miltenyi Biotec (2 mentions) Collagenase type xi, by Merck Group (2 mentions) Rmm csf, by BioLegend (2 mentions) Magnetic isolation kit, by Miltenyi Biotec (1 mentions) Counting beads, by BD (1 mentions) Fty720, by Merck Group (1 mentions) Easysep mouse neutrophil enrichment kit, by STEMCELL (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Cd31 fitc, by BioLegend (1 mentions) Fortessa x20, by BD (1 mentions) Totalreactive oxygen species ros assay kit, by Thermo Fisher Scientific (1 mentions) Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Facscanto 2, by BD (1 mentions) Glucose rpmi 1640, by Thermo Fisher Scientific (1 mentions) 70 μm cell strainer, by BD (1 mentions) Gallios flow cytometer, by Beckman Coulter (1 mentions)

11 protocols using astrios sorter

LEC-T Cell Migration Assay

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LECs were sorted by flow cytometry (Astrios sorter; Beckman Coulter) from Atg5^ΔProx1 and Atg5^WT stromal cell cultures and incubated (100,000 LECs/well) in 24-well plates coated as before. At cell confluence, cells were starved (in HBSS) or not starved overnight (250 µl). LEC culture medium was supplemented with 600 µl of RPMI, and transwell membrane (5-µm pores; Cornix) was added in each well. CD4⁺ T cells were purified from naive mice using a magnetic isolation kit (Miltenyi Biotec) and activated in vitro (anti-CD3 + anti-CD28 antibodies) for 3 d. Then, activated CD4⁺ cells were treated or not with FTY-720 (100 nM; Sigma-Aldrich) for the last 12 h, washed, and added (150,000 cells) in the upper part of the transwell. 4 h later, CD4⁺ T cell migration was analyzed in the bottom part by flow cytometry using counting beads (BD Bioscience). Results are expressed as ratio of CD4⁺ T cell migration compared with control group (unstarved Atg5^WT LECs with CD4⁺ T cells untreated with FTY-720).

Harlé G., Kowalski C., Dubrot J., Brighouse D., Clavel G., Pick R., Bessis N., Niven J., Scheiermann C., Gannagé M, & Hugues S. (2021). Macroautophagy in lymphatic endothelial cells inhibits T cell–mediated autoimmunity. The Journal of Experimental Medicine, 218(6), e20201776.

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Isolation and Characterization of Murine Neutrophils

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BM neutrophils were isolated with biotinylated mouse Ly6G Abs
(BioLegend, San Diego, CA, USA) and streptavidin magnetic beads (Miltenyi
Biotec) using MACS-column purification (Miltenyi Biotec, Auburn, CA, USA) for
ex vivo study. Purity of CD11b⁺Ly6G⁺cells was >95% following isolation. Hema3 staining confirmed
multi-lobulated nuclei, indicating mature neutrophil morphology. Findings in
Fig. 1A (Cd274expression) and Fig. 6B were validated
using neutrophils isolated by negative selection using the EasySep Mouse
Neutrophil Enrichment Kit from StemCell Technologies (Vancouver, BC, Canada). BM
monocytes were isolated by FACS as
CD11b⁺Ly6G⁻Ly6C⁺ cells (Astrios
sorter, Beckman Coulter, Brea, CA, USA). BM neutrophils
(CD11b⁺Ly6G⁺) were also isolated by FACS when BM
monocytes are compared. BMDMs or BMDCs were generated by culturing BM cells with
rmM-CSF (20 ng/ml, BioLegend) or rmGM-CSF (20 ng/ml, BioLegend), respectively.
Complete RPMI medium was used for all cell culture studies.

Deerhake M.E., Cardakli E.D, & Shinohara M.L. (2022). Dectin-1 signaling in neutrophils upregulates PD-L1 and triggers ROS-mediated suppression of CD4+ T cells. Journal of leukocyte biology, 112(6), 1413-1425.

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Isolation and Separation of Islet Cells

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Islets of Langerhans were isolated by standard procedure based on collagenase (type XI; Sigma) digestion of pancreas followed by Ficoll purification (Petrenko et al. 2017a (link)). Islet cells were gently dissociated by 0.05% trypsin (GIBCO) treatment, resuspended in KRB solution (pH 7.4, supplemented with 0.3% free fatty acid bovine serum albumin [BSA; Sigma], 1.4 mM glucose, 0.5 mM EDTA). α-Cell and β-cell populations were separated by flow cytometry fluorescence activated cell sorting (FACS; Astrios sorter [Beckman Coulter]) based on fluorescence wavelength and intensity, cell singlet nature, size, and viability as described previously (Petrenko et al. 2017b (link)).

Petrenko V., Stolovich-Rain M., Vandereycken B., Giovannoni L., Storch K.F., Dor Y., Chera S, & Dibner C. (2020). The core clock transcription factor BMAL1 drives circadian β-cell proliferation during compensatory regeneration of the endocrine pancreas. Genes & Development, 34(23-24), 1650-1665.

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Isolation and Culture of viECs

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On day 7 of the viEC differentiation, cells were dissociated with Accutase and neutralized with cold Stempro-34 SFM medium. Cells were spun at 1,000 rpm for 5 min and then washed with MACS buffer containing DPBS with 0.5% BSA and 2 mM EDTA. Cells were resuspended in 200 μL MACS buffer and co-stained for 30 min on ice with 10 μL pre-conjugated FITC CD31 (BioLegend) antibody and PE CD144 (BioLegend) antibody. Cells were rinsed once with MACS buffer and sorted on a Beckman Coulter Astrios Sorter at the Duke Flow Cytometry Shared Resource Facility. Cells were then replated on collagen-coated plates and cultured in viEC-conditioned medium during passage 0 directly after sorting. Media were changed every other day. viECs were routinely passaged at 80%–90% confluency using Accutase onto collagen-coated plates and continuously cultured in viEC medium consisting of Stempro-34 SFM medium with 10% HI-FBS, 50 ng/mL VEGF165, and 2 μg/mL heparin after passage 1. HGPS and normal viECs were used between passages 1 and 4.

Atchison L., Abutaleb N.O., Snyder-Mounts E., Gete Y., Ladha A., Ribar T., Cao K, & Truskey G.A. (2020). iPSC-Derived Endothelial Cells Affect Vascular Function in a Tissue-Engineered Blood Vessel Model of Hutchinson-Gilford Progeria Syndrome. Stem Cell Reports, 14(2), 325-337.

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Multicolor Flow Cytometry for Cell Phenotyping

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Live cell discrimination was accomplished using a LIVE/DEAD™
fixable dead cell stain kit (Invitrogen, Carlsbad, CA, USA) to stain single cell
suspensions prior to staining with fluorochrome-conjugated antibodies (Abs) (see
Supplemental Table
1 for detailed Ab information). Cells were analyzed by flow cytometry
using either BD Fortessa X20 or BD FACSCanto II analyzers (BD systems, Durham,
NC, USA) and results were analyzed using the FlowJo software. Flow cytometry
results were analyzed in all experiments using (1) an initial FSC-A/SSC-A gate
to eliminate low FSC-A/SSC-A debris, (2) a FSC-H/FSC-A gate to identify single
cells, and (3) Fixable Live/Dead stains to identify live cells prior to gating
for further analysis. Gating strategies are described in the text and specific
cell types were gated as neutrophils (CD11b⁺Ly6G⁺),
monocytes (CD11b⁺Ly6G⁻Ly6C^hi), cDCs
(CD11b⁺CD11c⁺, with MHC-II^hi for some
experiments), F4/80^hi macrophages
(CD11b⁺F4/80^hi), and CD4⁺ T cells
(CD3⁺CD4⁺). ROS production was assessed using a Total
Reactive Oxygen Species (ROS) Assay Kit (Invitrogen), in which cells are labeled
with a probe that increases in fluorescence upon interaction with ROS.
Fluorescence-activated single-cell sorting (FACS) was performed using an Astrios
sorter (Beckman Coulter, Brea, CA, USA), and at least one representative
post-sort sample was re-analyzed for purity during each sorting experiment.

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S1P Quantification in LEC Cultures

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LECs were sorted by flow cytometry (Astrios sorter; Beckman Coulter) from LEC/FRC cultures of Atg5^ΔProx1 and Atg5^WT LNs, and 100,000 cells/well were seeded in 12-well plates coated as before. At confluence, LECs were washed in PBS and cultured in RPMI (FBS free) containing 1% penicillin/streptomycin and 1% BSA. After 48 h, supernatants were harvested and centrifuged at 10,000 g for 10 min to eliminate cell debris. Quantification of S1P in supernatants was performed using the General Sphingosine-1-Phosphate (S1P) ELISA kit (MyBioSource) according to the manufacturer’s instructions.

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Isolation and FACS Purification of Islet Cells

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Islets of Langerhans were isolated by standard procedure based on collagenase (type XI; Sigma) digestion of the pancreas followed by Ficoll purification (Wojtusciszyn et al. 2009 (link)). Islet cells were gently dissociated by trypsin (GIBCO) resuspended in KRB solution (pH 7.4) supplemented with 0.3 % free fatty acid bovine serum albumin (BSA) (Sigma), 1.4 mM glucose, and 0.5 mM EDTA). α-Cell and β-cell populations were separated by flow cytometry FACS (Astrios sorter, Beckman Coulter) based on fluorescence wavelength and intensity and cell singlet nature, size, and viability (Supplemental Fig. S2).

Petrenko V., Saini C., Giovannoni L., Gobet C., Sage D., Unser M., Heddad Masson M., Gu G., Bosco D., Gachon F., Philippe J, & Dibner C. (2017). Pancreatic α- and β-cellular clocks have distinct molecular properties and impact on islet hormone secretion and gene expression. Genes & Development, 31(4), 383-398.

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Isolation and Sorting of Pancreatic Islet Cells

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Ins^dsRED2+/Ngn3^eGFP+ C57BL/6 mice (10–12 weeks old) were used for mouse pancreatic islet isolation. Islets were isolated via collagenase digestion, handpicked under a stereomicroscope, and maintained in 11.1 mmol/L glucose/RPMI 1640 (Thermo Fisher Scientific, Madrid, Spain) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA).⁴⁶ (link) Isolated islets were gently dissociated with trypsin (Gibco), resuspended in PBS supplemented with 25 mmol/L HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) and 2.5 mmol/L EDTA, and filtered through a 70-μm cell strainer (Falcon). β Cells (dsRED2⁺) and non-β cells (dsRED2⁻) were separated via fluorescence-activated cell sorting (FACS; Astrios Sorter [Beckman Coulter]) based on the fluorescence intensity and wavelength and the size of individual cells.

Zhao F., Liu X., Wang Z., Lang H., Zhang T., Wang R., Lin X., He D., Shi P, & Pang X. (2020). Novel Mouse miRNA Chr13_novelMiR7354-5p Improves Bone-Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells. Molecular Therapy. Nucleic Acids, 19, 1110-1122.

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Isolation and Culture of Murine Myeloid Cells

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BM neutrophils were isolated using MACS-column purification (Miltenyi Biotec) with biotinylated mouse Ly6G Abs (BioLegend) and streptavidin magnetic beads (Miltenyi Biotec) for ex vivo study. BM monocytes, were isolated by flow cytometry-sorting as CD11b⁺Ly6G⁻Ly6C⁺ cells (Astrios sorter, Beckman Coulter). When neutrophils were compared to monocytes, flow cytometry-sorting was used to isolate neutrophils as CD11b⁺Ly6G⁺. BMDMs and BMDCs were generated by culturing BM cells with rmM-CSF (20 ng/ml, BioLegend) and rmGM-CSF (20 ng/ml, BioLegend), respectively. Complete RPMI medium was used for all cell culture studies.

Deerhake M.E., Danzaki K., Inoue M., Cardakli E.D., Nonaka T., Aggarwal N., Barclay W.E., Ji R.R, & Shinohara M.L. (2021). Dectin-1 limits autoimmune neuroinflammation and promotes myeloid cell-astrocyte crosstalk via Card9-independent expression of Oncostatin M. Immunity, 54(3), 484-498.e8.

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Characterization of Cancer Stem Cell Markers

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The cultured cells were characterized using Beckman Coulter Gallios Flow Cytometer for cancer stem cell markers CD133 (APC), EPcam (PE), CD24 (Vioblue) and CD44 (FITC). They were also stained for the surface marker CD146 (APC). The two population of CD146 with low and high expression were sorted out using Beckman Coulter MofloAstrios Cell Sorter. The cells were sorted by staining the cells with anti-human CD146 antibody P1H12 for 1 h and then washed with PBS (PBS, no calcium, no magnesium Gibco™). The sorted cell populations were then incubated in CO₂ incubator for half an hour and then washed with PBS™/™ and used for further analysis.
CD146-high/low cells were sorted with an Astrios sorter (Beckman Coulter, Villepinte, France) according to the manufacturer instructions, and the threshold was established at 2 × 10⁴ for the MFI (Mean Fluorescence Intensity).

Sharma A., Joshkon A., Ladjimi A., Traboulsi W., Bachelier R., Robert S., Foucault-Bertaud A., Leroyer A.S., Bardin N., Somasundaram I, & Blot-Chabaud M. (2022). Soluble CD146 as a Potential Target for Preventing Triple Negative Breast Cancer MDA-MB-231 Cell Growth and Dissemination. International Journal of Molecular Sciences, 23(2), 974.

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