Moflo legacy

Manufactured by Beckman Coulter

Sourced in United States

The MoFlo Legacy is a flow cytometry system designed for high-performance cell sorting and analysis. It features a compact and modular design, allowing for customization to meet specific laboratory requirements. The MoFlo Legacy provides users with precise control over sample handling, data acquisition, and sorting parameters to facilitate accurate and efficient cell sorting.

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Lab products found in correlation

Shandon cytospin 4, by Thermo Fisher Scientific (3 mentions) Propidium iodide, by Thermo Fisher Scientific (3 mentions) Viobility 405 520 fixable dye, by Miltenyi Biotec (2 mentions) F4 80 antibody conjugated to alexa fluor 488, by BioLegend (2 mentions) Ly 6g antibody conjugated to apc cy7, by BioLegend (2 mentions) Tri reagent, by Molecular Research Center (2 mentions) Dispase, by Roche (2 mentions) Collagenase 1, by Worthington (2 mentions) Lsrii instrument, by BD (2 mentions) Moflo xdp, by Beckman Coulter (2 mentions) Agilent whole human genome oligo microarray, by Agilent Technologies (2 mentions) Cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions) Cd8 microbead, by Miltenyi Biotec (2 mentions) Agilent feature extraction software, by Agilent Technologies (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions) Anti cd3, by Beckman Coulter (1 mentions) Anti cd28, by Beckman Coulter (1 mentions) Alexa fluor 488 anti samhd1, by Euromedex (1 mentions) Rabbit anti samhd1, by Euromedex (1 mentions)

30 protocols using moflo legacy

CD4+ T-cell Activation and SAMHD1 Analysis

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Purified CD4⁺ T-lymphocytes from healthy individuals were separated from PBMCs using the CD4⁺ T-cell Isolation Kit II (Miltenyi Biotech) and stained with either 0.25 μmol CFSE or 0.5 μmol Cell Trace Violet (Invitrogen). Purified CD4⁺ T cells (1 × 10⁶) were cultured in RPMI-1640 (Life Technologies) containing l-glutamine, 10% fetal calf serum and antibiotics (penicillin and streptomycin) on a precoated 48-well plate with anti-CD3 (2 μg/ml) and anti-CD28 (2 μg/ml) (Beckman Coulter, Villepinte, France). After 72 h, cells were stained with Vioblue anti-CD3, APC-Vio770 anti-CD4 (Miltenyi Biotech) together with the Aqua Live/Death Vivid detection kit (Invitrogen). Samples were fixed and permeabilized using the FoxP3 permeabilization kit (eBioscience) and further stained with either Alexa Fluor 488 anti-SAMHD1 (a gift from O. Schwartz) or rabbit anti-SAMHD1 (Euromedex) followed by Alexa-Fluor 647 antirabbit antibody (Invitrogen). Fluorescence intensities were measured as described previously. Alternatively, CFSE-labeled cells were sorted according to CFSE fluorescence intensity using a MoFlo Legacy (Beckman Coulter) for mRNA quantification.

Ruffin N., Brezar V., Ayinde D., Lefebvre C., Wiesch J.S., van Lunzen J., Bockhorn M., Schwartz O., Hocini H., Lelievre J.D., Banchereau J., Levy Y, & Seddiki N. (2015). Low SAMHD1 expression following T-cell activation and proliferation renders CD4+ T cells susceptible to HIV-1. AIDS (London, England), 29(5), 519-530.

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Isolation and Purification of Scx-GFP Tendon Progenitors

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E14 embryos were harvested from pregnant Scx-GFP mice and staged [40 ] with Tufts University Institutional Animal Care and Use Committee approval. Limbs were isolated, minced, incubated under agitation at 200 rpm in 1% type II collagenase in PBS at 37°C for 45 minutes, and neutralized with GM. Cell suspensions were passed through a 40-μm cell strainer (BD Biosciences, San Jose, CA, USA), pelleted, washed in PBS, re-suspended in GM, plated at 1 × 10⁴ cells/cm², and cultured at 37°C and 5% CO₂. Three independent limb cell pools were harvested. Cells were trypsinized when 80% confluent and sorted on the basis of GFP signal using a MoFlo Legacy cell sorter (Beckman Coulter, Brea, CA, USA) at 488 nm excitation and collected by a 530/40 filter. TPCs were expanded to passage 1–2.

Brown J.P., Galassi T.V., Stoppato M., Schiele N.R, & Kuo C.K. (2015). Comparative analysis of mesenchymal stem cell and embryonic tendon progenitor cell response to embryonic tendon biochemical and mechanical factors. Stem Cell Research & Therapy, 6(1), 89.

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Allogeneic PBMC Proliferation Assay

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The responding cells (PBMCs) were first CFSE-labeled [5(6)-carboxyfluorescein diacetate N-succinimidyl ester; Sigma-Aldrich] and incubated at a ratio 1:1 (concentration of 1 × 10⁶ cells/ml) with stimulating allogeneic cells [Apo-cells or gamma-irradiated (20 Gy) PBMCs] at 37°C for 5 days. As a control, non-stimulated responding cells were cultivated in medium alone. The primary MLR was initiated by cultivating CD2– cells (CFSE-labeled), obtained from stimulating donor cells or non-labeled apoptotic cells, with allogeneic responding cells for 5 days. For the secondary stimulation, CD4-CD8 living cells from responding donor (CD4⁺, CD8⁺, CFSE⁻, 7AAD⁻) were sorted by fluorescence-activated cell sorting (FACS) (MoFlo Legacy, Beckman Coulter), then stained with cell proliferation dye eFluor450 (eBiosciences, France), and cultured with initial PBMCs from stimulating donor or third-party PBMCs at a ratio 1:1.

Pilon C., Stehlé T., Beldi-Ferchiou A., Matignon M., Thiolat A., Burlion A., Grondin C., Birebent B., Pirenne F., Rouard H., Lang P., Marodon G., Grimbert P, & Cohen J.L. (2019). Human Apoptotic Cells, Generated by Extracorporeal Photopheresis, Modulate Allogeneic Immune Response. Frontiers in Immunology, 10, 2908.

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Isolation and Expansion of GFP-Negative Cells

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Cells were detached using trypsin-EDTA, resuspended in DMEM containing 10% FBS, washed twice with PBS 1X by centrifugation (1200 rfc, 5 min), and finally resuspended in 1 mL of PBS containing 2% FBS at a cell concentration of 10⁶ cells/mL. Approximately 10⁶ events were analysed and naïve B16 and CT26 populations were used to delineate quadrants manually selecting survival, GFP-negative B16/CT26 cells. Selected cells were separated using a Beckman Coulter “MoFlo Legacy” cell sorter in 96-well plates and surviving cells were amplified by serial transfer to 48-well, 24-well, 60 mm, and 100 mm plates, and finally stored at −150°C.

Larrieux A, & Sanjuán R. (2022). Cellular resistance to an oncolytic virus is driven by chronic activation of innate immunity. iScience, 26(1), 105749.

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Single-cell sorting of pancreatic islet cells

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Pancreatic islets from Sst-Cre-GCaMP3 mice were dissociated into single cells by trypsin digestion and mechanical dissociation as described previously¹⁴ and filtered through a 30 μm filter to remove remaining clumps of cells.
Single cells were passed through a MoFlo Legacy (Beckman Coulter). GCaMP3- or RFP-positive cells were purified by combining several narrow gates (Supplementary Fig. 3). Forward and side scatter were used to isolate small cells and to exclude cell debris. Cells were then gated on pulse width to exclude doublets or triplets. GCaMP3-positive cells were excited with a 488 nm laser and the fluorescent signal was detected through a 530/40 nm bandpass filter (i.e. in the range 510-550 nm). RFP was excited with the 488 nm laser and the fluorescent signal was detected through a 580/30 nm bandpass filter (i.e. in the range 565-595nm). GCaMP3- or RFP-negative cells were collected in parallel.

Vergari E., Denwood G., Salehi A., Zhang Q., Adam J., Alrifaiy A., Wernstedt Asterholm I., Benrick A., Chibalina M.V., Eliasson L., Guida C., Hill T.G., Hamilton A., Ramracheya R., Reimann F., Rorsman N.J., Spilliotis I., Tarasov A.I., Walker J.N., Rorsman P, & Briant L.J. (2020). Somatostatin secretion by Na+-dependent Ca2+-induced Ca2+ release in pancreatic delta-cells. Nature metabolism, 2(1), 32-40.

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Isolation of Murine T Cell Subsets

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Purified cell populations were isolated from single-cell suspensions of leukocytes extracted from spleens and LNs. Briefly, WT cells were enriched by magnetic separation using a Pan T Cell Isolation Kit (Miltenyi Biotec), labeled with anti-Vγ4 (clone UC3-1OA6, 0.1 µg/10⁶ cells; BioLegend) or anti-TCRδ (clone GL3, 0.1 µg/10⁶ cells; eBioscience), sorted by flow cytometry, and incubated with anti-TCRβ (clone H57-597, 0.1 µg/10⁶ cells; eBioscience). Alternatively, WT cells were enriched by magnetic separation using a γδ T Cell Isolation Kit (Miltenyi Biotec). In the EAE model, Vγ4⁺TCRβ⁺ cells were enriched from spleens and draining LNs on day 10 after immunization with MOG and CFA. Distinct cell populations were sorted by flow cytometry using a FACSAria Fusion (BD Biosciences) or a MoFlo Legacy (Beckman Coulter).

Edwards S.C., Sutton C.E., Ladell K., Grant E.J., McLaren J.E., Roche F., Dash P., Apiwattanakul N., Awad W., Miners K.L., Lalor S.J., Ribot J.C., Baik S., Moran B., McGinley A., Pivorunas V., Dowding L., Macoritto M., Paez-Cortez J., Slavin A., Anderson G., Silva-Santos B., Hokamp K., Price D.A., Thomas P.G., McLoughlin R.M, & Mills K.H. (2020). A population of proinflammatory T cells coexpresses αβ and γδ T cell receptors in mice and humans. The Journal of Experimental Medicine, 217(5), e20190834.

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Insulin-positive and -negative cell sorting

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Human pancreatic islets of non-diabetic subjects were cultured with standard medium, or in the presence of IGFBP3 for 72 h as previously described (see “Pancreatic islets and Interventional studies”), and dissociated by trypsinization. In order to select insulin-positive and -negative populations, the cell fraction obtained was permeabilized using the Fixation and Permeabilization Solution Kit (554714, BD Biosciences) and stained with an APC-conjugated anti-insulin antibody (1:100, IC1417A, R&D Systems). Stained cells were flow-sorted using MoFlo Legacy (Beckman Coulter, Brea, CA) and analyzed by qRT-PCR.

D’Addio F., Maestroni A., Assi E., Ben Nasr M., Amabile G., Usuelli V., Loretelli C., Bertuzzi F., Antonioli B., Cardarelli F., El Essawy B., Solini A., Gerling I.C., Bianchi C., Becchi G., Mazzucchelli S., Corradi D., Fadini G.P., Foschi D., Markmann J.F., Orsi E., Škrha J J.r., Camboni M.G., Abdi R., James Shapiro A.M., Folli F., Ludvigsson J., Del Prato S., Zuccotti G, & Fiorina P. (2022). The IGFBP3/TMEM219 pathway regulates beta cell homeostasis. Nature Communications, 13, 684.

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Tracking Cell Line Dynamics with UHRF1 Modulation

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The CRC cells (HCT116 and RKO) stably expressing exogenous WT UHRF1, domain mutant UHRF1 and corresponding empty vector, were lentivirally transduced by the pLKO.3G-based vector (Addgene #14748) expressing scramble or endogenous UHRF1 specific shRNA with a GFP selection marker. The GFP positive cells were selected two days post transduction by MoFlo Legacy (Beckman) and mixed with the WT HCT116 or RKO cells. The percentage of GFP positive cells were recorded every 3 days from day 2 to day 14 with MoFlo Legacy, and normalized to the percentage at day 2.

Kong X., Chen J., Xie W., Brown S.M., Cai Y., Wu K., Fan D., Nie Y., Yegnasubramanian S., Tiedemann R.L., Tao Y., Yen R.W., Topper M.J., Zahnow C.A., Easwaran H., Rothbart S.B., Xia L, & Baylin S.B. (2019). Defining UHRF1 Domains That Support Maintenance of Human Colon Cancer DNA Methylation and Oncogenic Properties. Cancer cell, 35(4), 633-648.e7.

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CD8+ T cell Isolation from Spleen and Liver

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Single-cell suspensions from spleens and livers were stained with Viobility 405/520 fixable dye (Miltenyi), with PB-conjugated anti-CD8α (clone 53-6.7) and PE-conjugated anti-CD45.1 Abs. Live CD8⁺ CD45.1⁺ cells were sorted on a MoFlo Legacy (Beckman Coulter) cell sorter in a buffer containing PBS with 2% FBS. Cells were always at least 98% pure (data not shown).

Bénéchet A.P., De Simone G., Di Lucia P., Cilenti F., Barbiera G., Le Bert N., Fumagalli V., Lusito E., Moalli F., Bianchessi V., Andreata F., Zordan P., Bono E., Giustini L., Bonilla W.V., Bleriot C., Kunasegaran K., Gonzalez-Aseguinolaza G., Pinschewer D.D., Kennedy P.T., Naldini L., Kuka M., Ginhoux F., Cantore A., Bertoletti A., Ostuni R., Guidotti L.G, & Iannacone M. (2019). Dynamics and genomic landscape of CD8+ T cells undergoing hepatic priming. Nature, 574(7777), 200-205.

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Isolating Scx-GFP Tendon Cells from Mouse Limbs

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Scx-(green fluorescent protein) GFP-expressing tendon cells were isolated from limbs as previously described^{30 (link), 31 (link)}. Briefly, P7 and pregnant ScxGFP mice were sacrificed according to IACUC guidelines. E15 embryos were harvested from the pregnant mice and staged³², and limbs were harvested. ScxGFP-expressing cells were isolated from digested limbs of the litter via cell sorting by GFP signal (MoFlo Legacy, Beckman Coulter). Three independent P7 and E15 limb cell pools (litters) were harvested. Tendon cells were expanded to passages between 3 and 5 in growth medium (GM: high glucose Dulbecco’s Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S)) at 37°C and 5% CO₂ for experiments.

Li J., Schiele N.R., Stoppato M., Graybeal K.L., Nguyen P.K, & Kuo C.K. (2019). Embryonic and Postnatal Tendon Cells Respond Differently to IL-1beta. Annals of the New York Academy of Sciences, 1442(1), 118-127.

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