Macsquant vyb flow cytometer

Manufactured by Miltenyi Biotec

Sourced in Germany, United States, France

The MACSQuant VYB flow cytometer is a compact and versatile instrument designed for multicolor flow cytometry analysis. It features a violet (405 nm), blue (488 nm), and yellow-green (561 nm) laser configuration, providing multiple excitation wavelengths for the detection of a wide range of fluorescent markers. The instrument is capable of analyzing up to 14 parameters simultaneously and can process sample volumes ranging from 20 μL to 5 mL. The MACSQuant VYB is equipped with automated setup and quality control functionalities to ensure reliable and reproducible results.

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97 protocols using macsquant vyb flow cytometer

Salicylate-Induced Fluorescence Quantification

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Cultures inoculated from single colonies (2 ml; strains IE01 and IE02) were grown overnight in LB medium with and without salicylate (5 mM) at 37 °C and 170 rpm (to have uninduced and pre-induced cultures). Cultures were subsequently diluted 1:200 in M9 minimal medium with different concentrations of salicylate, and were grown for 4 h at the same conditions. Cultures were then transferred to Eppendorf tubes (1 ml). The fluorescence distribution of each sample was obtained with a flow cytometer MACSQuant VYB (Miltenyi Biotec) measuring yellow fluorescence (488 nm, 525 nm). Using delimited forward and side scatter ranges with the commercial software installed in the machine gated events. To calculate the normalized fluorescence, the background value was subtracted, and the resulting values were rescaled according to the mathematical model. The medians and standard deviations of the distributions were computed.

Rodrigo G., Bajić D., Elola I, & Poyatos J.F. (2017). Deconstructing a multiple antibiotic resistance regulation through the quantification of its input function. NPJ Systems Biology and Applications, 3, 30.

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Cell Proliferation and Cell Cycle Analysis

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Cell proliferation was evaluated using CellTiter 96AQueus One Solution (Promega, Madison, USA) and cell counting. For cell cycle analysis, NPM-ALK⁺ ALCL cells were washed with PBS and fixed overnight at 4°C in 70% ethanol. Cells were then washed with PBS supplemented with 0.1% BSA and incubated for 30 min with 10 μg/mL propidium iodide (Invitrogen, Waltham, USA) and 500 μg/mL of RNAse (Sigma-Aldrich, Saint Quentin Fallavier, France). Thereafter, cells were analyzed with a flow cytometer MACSQUANT VYB (Miltenyi, Paris, France).

Hoareau-Aveilla C., Quelen C., Congras A., Caillet N., Labourdette D., Dozier C., Brousset P., Lamant L, & Meggetto F. (2019). miR-497 suppresses cycle progression through an axis involving CDK6 in ALK-positive cells. Haematologica, 104(2), 347-359.

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Production and Quantification of rHAZV-eGFP Virus

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For production of viral stocks, rHAZV-eGFP virus^{24 (link)} was amplified in Huh-7.5 cells (MOI = 0.001). 1 h post-infection, media was changed after a PBS wash and 72 h post-infection, supernatant was harvested and clarified by centrifugation 5 min at 750 x g. Preparations of rHAZV-eGFP (termed HAZV in the text and figures) with titers of 10⁶ eGFP infection units (i.u.)/ml were used in this study.
For infection assays, Huh-7.5 cells were inoculated with serial dilutions of viral supernatant, corresponding to MOIs of 0.5 to 0.001, before PBS wash and medium change, 1 h post-infection. Level of infection was detected 16 h post-infection by quantification of eGFP positive cells by flow cytometry (MACSQuant® VYB Flow Cytometer; Miltenyi Biotec). Data were analyzed with FlowJo software (BD Biosciences). The viral titer was determined after selection of dilutions allowing a linear range of percentage of positive cells.

Ritter M., Canus L., Gautam A., Vallet T., Zhong L., Lalande A., Boson B., Gandhi A., Bodoirat S., Burlaud-Gaillard J., Freitas N., Roingeard P., Barr J.N., Lotteau V., Legros V., Mathieu C., Cosset F.L, & Denolly S. (2024). The low-density lipoprotein receptor and apolipoprotein E associated with CCHFV particles mediate CCHFV entry into cells. Nature Communications, 15, 4542.

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Inducible MDAR Memory Plasmid in E. coli

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Electrocompetent XL1-blue E. coli cells were cotransfected with the Arabinose trigger plasmid^{17 (link)} and MDAR memory plasmid or standard memory plasmid,^{17 (link)} respectively. Cell culture experiments were
carried out at 28 °C basically as described in Ullrich et al.^{30 (link)} Single colonies were used for the inoculation
of overnight cultures in LB medium supplemented with 25 μg/mL
kanamycin, 100 μg/mL ampicillin, 300 μM ZnSO₄, and 0.2% glucose to generate a stable OFF-culture. After 24 h,
the ZnSO₄ concentration was decreased to 10 μM by
exchanging the medium. Induction of the ccrM gene expression was performed
by replacing the glucose-containing medium with arabinose (0.02% (w/v))
containing medium. The EGFP expression was evaluated via flow cytometry
as described in Ullrich et al.^{30 (link)} using
a MACSQuant VYB Flow cytometer (Miltenyi Biotec). Data analysis was
performed with FLOWJO V10.8.0 software (FlowJo BD, Heidelberg Germany).

Klingel V., Graf D., Weirich S., Jeltsch A, & Radde N.E. (2022). Model-Based Design of a Synthetic Oscillator Based on an Epigenetic Methylation Memory System. ACS Synthetic Biology, 11(7), 2445-2455.

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Macrophage Differentiation and TNFα Evaluation

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To promote differentiation into macrophages, isolated human monocytes were cultured in the presence of human recombinant MCSF (20 ng/mL; Peprotech) in RPMI 1640 (Life Technologies, Carlsbad, CA, USA) containing FBS (10%; Life Technologies), l-glutamine (2 mM), and penicillin-streptomycin (Life Technologies) at 37 °C in 5% CO₂. human recombinant MCSF was repeatedly added every 2 days after the initiation of the culture. At day 6, generated macrophages were treated with LPS (10 ng/mL) and incubated for 18 h in the presence or absence of various concentrations of MI-2. The culture supernatants were collected and kept at −80 °C until analysis, and TNFα levels in the culture supernatants were measured using the Human TNFα ELISA MAX^TM Deluxe Kit following the manufacturer’s procedure (BioLegend). To identify human macrophages, cells were stained with antibodies against human CD68 and TNFα (BioLegend) using the BioLegend Intracellular Staining Kit according to the manufacturer’s instructions prior to analysis by flow cytometry. Staining data were analysed on a MACSQuant®VYB flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany).

Lee C.H., Bae S.J, & Kim M. (2017). Mucosa-associated lymphoid tissue lymphoma translocation 1 as a novel therapeutic target for rheumatoid arthritis. Scientific Reports, 7, 11889.

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DNA Synthesis Measurement in THP-1 Cells

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DNA synthesis was assessed in THP-1 cells using previously described methods. Briefly, THP-1 cells were stimulated with PMA for 7 days or grown in suspension culture. Media was replaced with complete RPMI containing 10 μm 5-bromo-2′-deoxyuridine (Sigma). Cells were grown in the presence of BrdU for 1 or 8 h, harvested, and fixed by addition of 70% ice-cold ethanol. Cells were permeabilized by addition of 0.5% Triton X-100 and DNA was denatured with 2 n HCl, neutralized, washed, and stained with an anti-BrdU antibody (Cell Signaling Technology; Bu20a) diluted 1:50 in a solution of 1× PBS, 1% BSA, and 0.1% Tween 20. Cells were washed then stained with Alexa Fluor 488 goat anti-mouse antibody (Invitrogen) diluted 1:50. Cells were washed and analyzed using a MACSQuantVYB flow cytometer (Miltenyi Biotec) and data were analyzed using MACSQuantify software (Miltenyi Biotec).

Goetze R.W., Kim D.H., Schinazi R.F, & Kim B. (2017). A CRISPR/Cas9 approach reveals that the polymerase activity of DNA polymerase β is dispensable for HIV-1 infection in dividing and nondividing cells. The Journal of Biological Chemistry, 292(34), 14016-14025.

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Multicolor Flow Cytometry of Mouse Splenocytes

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At specific time points following mouse inoculation with various agents, mice were euthanized by CO2 inhalation followed by rapid dissection of the spleen. Mouse spleens were minced on ice and a cellular suspension was separated from tissue using a 40 μm cell strainer. Red blood cells were removed by rapid exposure of mouse splenocytes to ACK lysis buffer followed by washing with FACS buffer (PBS with 2% fetal bovine serum and 2 mM EDTA). Single cell suspensions of mouse splenocytes were first incubated with rat anti-mouse CD16/CD32 (BD Pharmingen #553142) on ice for 15 min, followed by incubation with fluorophore-conjugated antibodies and/or TNF-α conjugated liposomes encapsulating Alexa-594 for 10 min on ice covered by aluminum foil. The cells were washed twice with 200 μl FACS buffer, resuspended in FACS buffer, and fixed in the presence of 2% PFA at 22°C for 1 hour before data acquisition on a Miltenyi MACSQuant VYB flow cytometer equipped with 3 spatially-separated lasers (405, 488 and 561 nm) and 10 detection channels. A minimum of 100,000 events were collected for all experiments. The fluorophore-conjugated antibodies were pacific blue rat anti-mouse CD19 (Biolegend #115526), FITC rat anti-mouse GL7 antigen (Biolegend #144603). The data were analyzed using FlowJo (BD).

Chen Z., Wholey W.Y., Najafabadi A.H., Moon J.J., Grigorova I., Chackerian B, & Cheng W. (2019). Self Antigens Displayed on Liposomal Nanoparticles above a Threshold of Epitope Density Elicit Class-switched Autoreactive Antibodies Independent of T-cell Help. Journal of immunology (Baltimore, Md. : 1950), 204(2), 335-347.

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Isolation and Activation of Splenic CD8+ T Cells

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Splenic CD8⁺ T cells were isolated by negative selection using the MojoSort Mouse CD8⁺ T Cell Isolation Kit (Biolegend) following the manufacturer's instructions. Isolated cells were plated at 2×10⁶ per mL in RPMI 1640 containing 2.0 mM L-glutamine and 25 mM HEPES, supplemented with 10 mM Sodium Pyruvate, nonessential amino acids, 100 U/ml penicillin/streptomycin, 55 μM β-ME, and 10% fetal calf serum (complete media). Cells were stimulated with 1 μg/mL ionomycin (Invivogen) and 1 ng/mL phorbol myristate acetate (PMA, Invivogen) for 7 days, with complete media supplemented with 200 U/mL recombinant human IL-2 added on days 3 and 6. Following stimulation, cells were resuspended at 5×10⁵ per mL in RPMI 1640 supplemented with 0.1% bovine serum albumin, and were incubated at 4°C for 60 min with or without 100 nM (±)-AMG 487 (R&D Systems). Cells were then plated in the top well of 96 well transwell plate (3 μm polycarbonate membrane pore, Corning). The bottom well of the plate contained RPMI 1640 supplemented with 0.1% BSA, either with or without recombinant mouse CXCL9 or CXCL10 (Biolegend). Cells were allowed to migrate for 1 hr at 37°C, prior to data collection with a MACSQuant VYB flow cytometer (Miltenyi Biotech) and analysis using FlowJo version 10.

de Mingo Pulido Á., Gardner A., Hiebler S., Soliman H., Rugo H.S., Krummel M.F., Coussens L.M, & Ruffell B. (2018). TIM-3 regulates CD103+ dendritic cell function and response to chemotherapy in breast cancer. Cancer cell, 33(1), 60-74.e6.

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Cell Proliferation Kinetics by Flow Cytometry

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HAP1 cells were labeled using the CellTrace™ Violet (CTV) Cell Proliferation Kit (Invitrogen) according to the manufacturer's instructions. A 2 μl aliquot of CTV was incubated with 2 × 10⁶ HAP1 cells in 2 ml of PBS for 20 min at 37°C. To neutralize the unbound CTV, 8 ml of DMEM was added. After two washes with PBS, every cellular clone was plated in a 6-well plate at 3 × 10⁵ cells per well and grown with or without selenium supplementation. Cells were harvested at the indicated time points and analyzed by flow cytometry all at once at the end of the kinetics. Flow cytometry was performed on a MACSQuant® VYB Flow cytometer (Miltenyi Biotec) and the analysis was done with FlowJo software (Treestar, Ashland, OR, USA). The mean fluorescence intensity (MFI) of each condition was plotted as a function of time. Kinetic curves were fitted with y = a.exp(−k.t), where the doubling time t_1/2 = [ln(2)/k], and the represented time necessary for a 50% decrease in the MFI signal.

Vindry C., Guillin O., Wolff P., Marie P., Mortreux F., Mangeot P.E., Ohlmann T, & Chavatte L. (2023). A homozygous mutation in the human selenocysteine tRNA gene impairs UGA recoding activity and selenoproteome regulation by selenium. Nucleic Acids Research, 51(14), 7580-7601.

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Flow Cytometric Analysis of Lymphocytes

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Unless otherwise noted, flow cytometry analysis and fluorescent-activated cell sorting of all in vitro and ex vivo lymphocytes were prepared using the procedures outlined. Briefly, cultured cells on tissue culture plates and primary cells from lymphoid organs were prepared as single cell suspensions, incubated in 2.4G2 Fc blocking solution, stained with respective surface cell markers as indicated (see Supplementary file 1, Key Resources Table), resuspended in HBH, filtered through a 40 μm nylon mesh, and analyzed using a benchtop MacsQuant VYB flow cytometer (Miltenyi Biotec, Auburn, CA) or sorted with Sony Synergy Sorter (Sony Biotechnology, Inc, San Jose, CA). Both instruments contain capabilities to detect mCherry fluorescence by 561 nm laser excitation. All antibodies used in these experiments are standard, commercially available monoclonal reagents widely established to characterize immune cell populations in the mouse; details are given in Supplementary file 1. Acquired flow cytometry data were all analyzed with FlowJo software (Tree Star).

Ng K.K., Yui M.A., Mehta A., Siu S., Irwin B., Pease S., Hirose S., Elowitz M.B., Rothenberg E.V, & Kueh H.Y. (2018). A stochastic epigenetic switch controls the dynamics of T-cell lineage commitment. eLife, 7, e37851.

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