Moflo sorter

Manufactured by Beckman Coulter

Sourced in United States

The MoFlo sorter is a high-performance cell sorting instrument designed for use in flow cytometry applications. The core function of the MoFlo sorter is to rapidly and accurately sort and isolate specific cell populations from complex biological samples.

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Lab products found in correlation

Primary ov6 antibody mouse igg1, by R&D Systems (2 mentions) Mini macs cell sorter kit, by Miltenyi Biotec (2 mentions) Apc conjugated ov6 antibody, by R&D Systems (2 mentions) Gallios flow cytometer, by Beckman Coulter (2 mentions) Lsrfortessa, by BD (2 mentions) Halo tag r110 direct ligand, by Promega (1 mentions) Cd8α apc cy7, by BD (1 mentions) E cadherin pe, by BioLegend (1 mentions) Cd11c apc, by BD (1 mentions) Cd11b pe, by BD (1 mentions) Pentr pter, by Addgene (1 mentions) Propidium iodide, by BioLegend (1 mentions) Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions) Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Anti cd11c apc clone b ly6, by BD (1 mentions) Cd33 apc, by Beckman Coulter (1 mentions) Anti cd33 fitc, by Beckman Coulter (1 mentions) Anti cd1d pe, by BD (1 mentions) Anti cd1a pe, by BD (1 mentions)

Close spelling variants of this product

MoFlo Astrios cell sorter (159 citations) MoFlo cell sorter (126 citations) MoFlo XDP sorter (47 citations) MoFlo high-speed cell sorter (34 citations) MoFlo XDP High-Speed Cell Sorter (22 citations) MoFlo™ XDP High-Performance Cell Sorter (12 citations) MoFlo flow sorter (8 citations) MoFlo High-Performance Cell Sorter (8 citations) MoFlo Astrios EQ FACS-sorter (3 citations) MoFlo high-speed sorter (3 citations)

20 protocols using moflo sorter

Enrichment and Characterization of OV6+ Cells

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Cells were labelled with primary OV6 antibody (mouse IgG1; R&D Systems, Minneapolis), magnetically tethered to rat anti-mouse IgG1 microbeads, and sorted with a Mini-MACS™ Cell Sorter Kit (Miltenyi Biotec, CA). All of the procedures were following the manufacturer’s instructions. The sorted cells were evaluated by flow cytometry analysis. The flow cytometry was performed with MoFlo Sorter (Beckman, Brea, CA) using an APC-conjugated-OV6 antibody (R&D Systems, Minneapolis) and following manufacturer’s instruction.

Zhao Y., Lu Q., Li C., Wang X., Jiang L., Huang L., Wang C, & Chen H. (2019). PRMT1 regulates the tumour-initiating properties of esophageal squamous cell carcinoma through histone H4 arginine methylation coupled with transcriptional activation. Cell Death & Disease, 10(5), 359.

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Enrichment of MLL^Dim Cells

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To generate Halo-tagged MLL^Dim cells, HEK293 cells were transfected with pFENHK-MLL plasmid with Polyethylenimine. 2 days later, the transfected cells were selected with 400 ng/ml G418 for 3 weeks and stained with HaloTag R110Direct ligand (Promega). Cells were then sorted according to the Halo-tag signals with a MoFlo sorter (Beckman Coulter) as MLL^Negative, MLL^Dim, MLL^Mid, and MLL^High. The sorted cells were grown in DMEM with 10% FBS and selected with 400 ng/ml G418. 2 weeks later, the cells were resorted according to HaloTag signals. The MLL^Dim were further sorted two more times.

Liang K., Volk1 A.G., Haug J.S., Marshall S.A., Woodfin A.R., Bartom E.T., Gilmore J.M., Florens L., Washburn M.P., Sullivan K.D., Espinosa J.M., Cannova J., Zhang J., Smith E.R., Crispino J.D, & Shilatifard A. (2017). Therapeutic targeting of MLL degradation pathways in MLL-rearranged leukemia. Cell, 168(1-2), 59-72.e13.

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Isolation and Sorting of Immune Cells

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Splenocytes were prepared from 12-week mice, erythrocytes were removed by incubation in lysis buffer (1:9 v/v 0.17M Tris: 0.16 M ammonium chloride) for 5 min and enriched splenocytes were resuspended in 2% FBS and 1 mM EDTA in Hank’s Balanced Salt Solution. For isolation of tumour lineages, dissected gp130^F/F gastric tumours were chopped into ~3–4mm³ pieces and disaggregated non-enzymatically by incubation in dissociation buffer (5% FBS, 1 mM dithiothreitol, 1 mM EDTA in PBS) for 1 h at 37 °C with agitation. Digested tissue pieces were passed through a 70-μm strainer, and the cells were resuspended in 2% FBS and 2 mM EDTA in Hank’s Balanced Salt Solution. Splenocytes and gastric tumour cells were stained with CD11c-APC (1:500), CD8α-APC-Cy7 (1:500), CD45R/B221-FITC (1:500), CD11b-PE (1:500), Gr-1 (Ly6-G/C)-PerCP-Cy5.5 (1:300) (all from BD Biosciences) and NK1.1-brilliant violet 421 (1:300) E-cadherin-PE (1:300) (from BioLegend, San Diego, CA, USA). Cells were sorted (at low pressure with a 100-μm nozzle) on a MoFlo sorter (Beckman-Coulter, Brea, CA, USA). Cells were not cultured in the period between isolation and sorting.

Kurklu B., Whitehead R., Ong E., Minamoto T., Fox J., Mann J., Judd L., Giraud A, & Menheniott T. (2014). Lineage-specific RUNX3 hypomethylation marks the preneoplastic immune component of gastric cancer. Oncogene, 34(22), 2856-2866.

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Identification of SARS-CoV-2 Specific T Cells

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PBMCs from 2 SARS-CoV-2 convalescent donors were expanded with LLL and M1 aAPCs for 14 days; stained with CD3, CD8, L/D, and LLL and M1 tetramers; and sorted on a MoFlo sorter (Beckman Coulter) Three fractions were collected: (a) double staining with LLL⁺ and M1⁺ tetramer; (b) LLL⁺ tetramer only; and (c) M1⁺ tetramer only.

Chaisawangwong W., Wang H., Kouo T., Salathe S.F., Isser A., Bieler J.G., Zhang M.L., Livingston N.K., Li S., Horowitz J.J., Samet R.E., Zyskind I., Rosenberg A.Z, & Schneck J.P. (2022). Cross-reactivity of SARS-CoV-2– and influenza A–specific T cells in individuals exposed to SARS-CoV-2. JCI Insight, 7(18), e158308.

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Generating shRNA Targeting Canine Hepsin and Prostasin

Cited 1 time

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Sfold (http://sfold.wadsworth.org/cgi-bin/index.pl) and siDESIGN center (http://www.thermoscientificbio.com/design-center/) were used to identify siRNAs targeting canine hepsin and prostasin. Selected oligos (sense sequence) are listed in Table 2. Oligonucleotides for shRNA strategy were designed combining the siRNA sequence (sense) followed by a loop (sequence 5’-3’: TTCAAGAGA), the reverse complement of the siRNA sequence (antisense) and RNA polymerase III terminator (sequence 5’-3’: TTTTTTGGAA). Oligos were phosphorylated, annealed and inserted in pENTR/pTER+, a gift from Eric Campeau (plasmid # 17453, Addgene, Cambridge, MA) (Campeau et al., 2009 (link)). MDCK cells stably expressing uromodulin were co-transfected with pENTR/pTER+ vector and EGFP-expressing vector pcDNA3x(+)MyEGFP (Life Technologies). Cells were collected 24 hr after transfection and sorted using MoFlo sorter (Beckman Coulter, Brea, CA). Recovered EGFP-positive cells were maintained in culture for 48 hr.

10.7554/eLife.08887.027

Table 2.

Oligonucleotide sequences used to generate shRNAs.

DOI:http://dx.doi.org/10.7554/eLife.08887.027

Target gene	Target position from ATG codon	siRNA oligo (5’-3’)
HPN_siRNA#1	122-140	CCTTCCTACTCAAGAGTGA
HPN_siRNA#2	1195-1213	TGGATCTTCCAGGCCATAA
PRSS8_siRNA#1	258-276	GAAGGAAGACTATGAGGTA
PRSS8_siRNA#2	593-611	TGTACAACATCAACGCTAA
Control siRNA	NA	TAGTGAGATTCGTTAAGAT

Brunati M., Perucca S., Han L., Cattaneo A., Consolato F., Andolfo A., Schaeffer C., Olinger E., Peng J., Santambrogio S., Perrier R., Li S., Bokhove M., Bachi A., Hummler E., Devuyst O., Wu Q., Jovine L, & Rampoldi L. (2015). The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin. eLife, 4, e08887.

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Isolation and Osteoclastogenesis of Murine Myeloid Progenitors

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To obtain lineage-negative CD11b⁺ and CD11b^- cells, WBM cells were red cell-lysed, first labeled with biotin-conjugated lineage antibodies and then incubated with anti-biotin magnetic microbeads to deplete lineage cells. Anti-CD11b microbeads was subsequently used to separate CD11b⁺ from CD11b^- cells. These MACS-purified cells were subjected to osteoclastogenesis assay as routinely done for WBMs or bone marrow macrophages, except they were seeded at a much lower series of density to obtain the optimal number of multinucleated osteoclast formation. MDSC isolation kit was used to obtain bone marrow cells that immuno-phenotypically resembled MDSCs.
For isolation of LSK HSCs, CMPs and GMPs, WBM cells were labeled with FACS antibodies as described above for progenitor analysis and sorted by either Sony Synergy (Sony Biotechnology, San Jose, CA, USA) or MoFlo sorter (Beckman Coulter, Indianapolis, IN, USA). These progenitor cells were sorted into ice-old culture alpha MEM supplemented with 20% FBS to maximize their survival rate. After sorting, cell counts were determined after resuspending cell pellets in a small volume of culture media before plating on 96-well microplates for osteoclastogenesis assay.

Chen T.H., Swarnkar G., Mbalaviele G, & Abu-Amer Y. (2015). Myeloid lineage skewing due to exacerbated NF-κB signaling facilitates osteopenia in Scurfy mice. Cell Death & Disease, 6(4), e1723-.

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Single-cell RNA Extraction Workflow

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At 6 h p.i., cells were harvested, washed, stained with Propidium Iodide (Biolegend) and resuspended in FACS buffer containing PBS, 0.3% (v/v) bovine serum albumin (BSA, Sigma) and 2 mM EDTA (Invitrogen). Samples were immediately acquired on a MoFlo sorter (Beckman Coulter) applying fluorescence minus one control to adjust compensation and sorting gates. The accuracy of a high purity single-cell sorting was confirmed using beads and cells. In total, 20,000 live single cells were sorted into Eppendorfs containing 10 µL of PBS supplemented with 40 U/µL of RiboLock RNase inhibitor (ThermoFisher), spun down, and immediately processed for RNA extraction.

Aulicino A., Antanaviciute A., Frost J., Sousa Geros A., Mellado E., Attar M., Jagielowicz M., Hublitz P., Sinz J., Preciado-Llanes L., Napolitani G., Bowden R., Koohy H., Drakesmith H, & Simmons A. (2022). Dual RNA sequencing reveals dendritic cell reprogramming in response to typhoidal Salmonella invasion. Communications Biology, 5, 111.

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Flow cytometric analysis of CD1 expression

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CD1 expression was determined using mAbs anti–CD1a-PE (clone HI149; BD), anti–CD1b-PE (clone 0249; Santa Cruz Biotechnology, Inc.), anti–CD1c-PE (clone L161; Santa Cruz Biotechnology, Inc.), and anti–CD1d-PE (clone 42.1; BD). Leukemia blasts were identified using anti–CD19-FITC or CD19-PerCPCy5.5 (both clone HIB19) and anti–CD14-FITC (clone HCD14; all from BioLegend); anti–CD3-FITC (clone UCHT1), anti–CD45-APC (clone HI30), and anti–CD117-PerCpCy5.5 (clone 2B8; all from BD); anti–CD7-PC5 (clone 8H8.1), anti–CD10-PC5 (clone ALB1), anti–CD33-FITC or CD33-PC5 or anti–CD33-APC (all clone D3HL60-251), anti–CD34-FITC or CD34-PC5 or anti–CD34-APC (all clone 581), and anti–CD45-PC7 (clone J33; all from Beckman Coulter). cDCs were sorted using anti-CD64 FITC (clone 22; Beckman Coulter) and anti–CD11c-APC (clone B-ly6; BD). Samples were acquired on CyAn ADP (Dako) or FACSCanto (BD) flow cytometers. Cell sorting experiments were performed using a MoFlo sorter (Beckman Coulter). Dead cells were excluded on the basis of propidium iodide and DAPI staining. All data were analyzed using FlowJo software (Tree Star). Relative fluorescent intensity (RFI) of CD1c expression on leukemia cells was calculated dividing the mean CD1c fluorescence intensity by the mean fluorescence intensity of the corresponding isotype control mAb.

Lepore M., de Lalla C., Gundimeda S.R., Gsellinger H., Consonni M., Garavaglia C., Sansano S., Piccolo F., Scelfo A., Häussinger D., Montagna D., Locatelli F., Bonini C., Bondanza A., Forcina A., Li Z., Ni G., Ciceri F., Jenö P., Xia C., Mori L., Dellabona P., Casorati G, & De Libero G. (2014). A novel self-lipid antigen targets human T cells against CD1c+ leukemias. The Journal of Experimental Medicine, 211(7), 1363-1377.

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Western Blot Analysis of Tsc1-T-KO CD4 T Cells

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Western blot analysis was performed as previously described (26 (link)). Briefly, WT and Tsc1-T-KO CD4 T cells were sorted from splenocytes with a Moflo sorter (Beckman Coulter). Cell lysates of 1 x10⁶ cells in 1% Nonidet P‐40 Lysis solution (1% Nonidet‐40, 150 mM NaCl, and 50 mM Tris, pH 7.4) with freshly added protease and phosphatase inhibitors were subjected to immunoblotting analysis with indicated antibodies. Anti-Tsc1 (D43E2, #6935), anti-phospho-S6 S235/236 (2F9, #4856), anti-phospho-4E-BP1 T37/46 (236B4, # 2855), anti-4E-BP1 (53H11, #9644), and anti-Akt1 (C73H10, #2938) antibodies were purchased from Cell Signaling Technology.

Zhang S., Li L., Xie D., Reddy S., Sleasman J.W., Ma L, & Zhong X.P. (2021). Regulation of Intrinsic and Bystander T Follicular Helper Cell Differentiation and Autoimmunity by Tsc1. Frontiers in Immunology, 12, 620437.

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Flow Cytometry Staining and Sorting

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Cells were washed twice in PBS with 2% BSA and stained with primary antibody for 45 minutes at 4°C in the dark. Cells were then washed and centrifuged twice at 500g and incubated with secondary antibody for an additional 30 minutes. After an additional centrifugation cells were stored on ice until fluorescence activated cell sorting (FACS) analysis, which was performed on a Becton Dickinson LSR Fortessa. Data were analyzed using FlowJo version 8.8.6 (Treestar Inc). DAPI staining and FSC-A and SSC-A gating were used to exclude dead cells and FSC-W and SSC-W gating was applied to exclude doublets. When needed, cell sorting was performed on a Beckman Coulter MoFlo sorter.

Ceppi P., Hadji A., Kohlhapp F.J., Pattanayak A., Hau A., Liu X., Liu H., Murmann A.E, & Peter M.E. (2014). CD95 and CD95L promote and protect cancer stem cells. Nature communications, 5, 5238.

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