Macsquant analyzer

Manufactured by Miltenyi Biotec

Sourced in Germany, United States, United Kingdom, Japan, France, Italy, Canada, Austria

The MACSQuant Analyzer is a flow cytometry instrument designed for multi-parameter analysis of cells and particles. It provides high-throughput data acquisition and analysis capabilities, enabling researchers to efficiently quantify and characterize various cell populations. The MACSQuant Analyzer features advanced optics, automated sample handling, and intuitive software for data processing and visualization.

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Lab products found in correlation

643 protocols using macsquant analyzer

Cell Viability, Apoptosis, and Cell Cycle Analysis

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To test cell viability, cells were plated at a density of 5 × 10⁴ viable cells/ml and treated. Upon incubation, cells were labelled with propidium iodide (PI) solution (2 µg/ml) (Millipore Sigma, Darmstadt, Germany) and the count of viable cells (PI negative) was performed using the flow cytometer MACSQuant Analyzer (Miltenyi Biotec).
For cell cycle analyses, cells were fixed in 70% ethanol and stained with 2 µg/ml PI. Cell cycle distribution was measured by flow cytometry (MACSQuant Analyzer).
For assessment of apoptosis cells were stained with Annexin V and PI (Annexin V-FITC Kit, Miltenyi Biotec) following the manufacturer’s instructions and analysed by flow cytometry (MACSQuant Analyzer). Mitochondrial membrane depolarisation was assayed using the fluorescent probe tetramethylrhodamine ethyl ester-TMRE (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions and measured by flow cytometry (MACSQuant Analyzer).
All data were analysed using the MACSQuantify software version 2.6 (Miltenyi Biotec).

Magni M., Biancon G., Rizzitano S., Cavanè A., Paolizzi C., Dugo M., Corradini P, & Carniti C. (2019). Tyrosine kinase inhibition to improve anthracycline-based chemotherapy efficacy in T-cell lymphoma. British Journal of Cancer, 121(7), 567-577.

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Isolation of Uveitic T Cells from Mouse Eyes

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To isolate T cells from eyes of uveitic mice, enucleated eyes were first trimmed of external tissue and then the globe was opened along the limbus to remove the lens. The remaining tissue was minced in HL-1 media (Lonza, Walkersville, MD). Cell/tissue separation was accomplished with 1.0 mg/ml collagenase D treatment (Roche, Indianapolis, IN) for 40 minutes at 37°C. After washing, tissue was dispersed through a 40 µm strainer, resuspended in PBS + 2% FBS + 2mM EDTA sorting buffer and then stained. For lymph nodes (LN), tissue was dispersed through a 40 µm strainer and then resuspended in sorting buffer. Cell populations were analyzed using a BD FACS Calibur (Becton Dickinson, San Jose, CA) and MACSQuant Analyzer (Miltenyi Biotec, Auburn, CA) or were sorted on a FACS Aria II (BD) to 99% purity. To determine the number of Tregs present in eyes of mice months after EAU challenge, eye cells were incubated with CD4⁺ isolation beads (Miltenyi Biotec) prior to staining and then passed through a magnetic column that was integrated into a MACSQuant Analyzer. Since the frequency of Tregs at these late time points was expected to be low, utilization of the column permitted the enrichment of the cells before analysis. All data were analyzed using FlowJo (TreeStar, Ashland, OR).

Silver P., Horai R., Chen J., Jittayasothorn Y., Chan C.C., Villasmil R., Kesen M.R, & Caspi R.R. (2015). Retina-specific T regulatory cells bring about resolution and maintain remission of autoimmune uveitis. Journal of immunology (Baltimore, Md. : 1950), 194(7), 3011-3019.

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Immunophenotyping of ECAR-armed T Cells

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Isolated T cells were stained with fluorochrome-labeled mabs directed against human CD4/VioBlue (Miltenyi Biotec, clone VIT4), CD3/PE-Cy7 (Biolegend, San Diego, USA, clone UCHT1), CD8/APC (BD Bioscience, clone RPA-T8), CD27/PE (BD Bioscience, clone M-T271) and CD62L/PacificBlue (Biolegend, clone DREG-56). For detection of ECAR surface expression, T cells were incubated with anti-La mab 5B9 [16] and subsequently stained with PE-labeled goat anti-mouse IgG (Beckmann Coulter, Krefeld, Germany). Samples were analyzed using the MACSQuant Analyzer and the MACSQuantify software (Miltenyi Biotec). In order to assess expansion rates of ECAR armed T cells, absolute T cell numbers were quantified using a MACSQuant Analyzer and MACSQuantify software (Miltenyi Biotec) as described elsewhere [25] (link).

Cartellieri M., Koristka S., Arndt C., Feldmann A., Stamova S., von Bonin M., Töpfer K., Krüger T., Geib M., Michalk I., Temme A., Bornhäuser M., Lindemann D., Ehninger G, & Bachmann M.P. (2014). A Novel Ex Vivo Isolation and Expansion Procedure for Chimeric Antigen Receptor Engrafted Human T Cells. PLoS ONE, 9(4), e93745.

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Mitochondrial Characterization in Multiple Myeloma

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MM cells were washed three times with Cell Staining Buffer (BioLegend) and resuspended in their respective cell culture media with 100 nM MitoTracker Green (Invitrogen, Cat. No. M7514), and incubated for 30 min at 37 °C. Cells were washed three times with Cell Staining Buffer and resuspended in Cell Staining Buffer (BioLegend) prior to flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec). To characterize mitochondrial membrane potential, MM cells were washed three times with Cell Staining Buffer (BioLegend) and resuspended in tetramethylrhodamine ethyl ester (TMRE) buffer (Cayman Chemicals) containing 100 nM TMRE (Cayman Chemicals, Cat. no. 701310) and incubated for 30 min at 37 °C. Cells were pelleted and resuspended in TMRE buffer and subjected to flow cytometry on a MACSQuant Analyzer (Miltenyi Biotec). For mitochondrial superoxide measurements, ATCC MM.1S cells were washed three times with Cell Staining Buffer (BioLegend) and stained with 5 μM MitoSOX^™ Red (Invitrogen, Cat. No. M36008) in Hank’s balanced salt solution with calcium and magnesium (HBSS/Ca²⁺/Mg²⁺, Giboco, 14025–092) for 10 min at 37 C. Cells were washed three times with warm HBSS/Ca²⁺/Mg²⁺ and resuspended in HBSS/Ca²⁺/Mg²⁺ before analysis by flow cytometry.

Murphy C.S., DeMambro V.E., Fadel S., Fairfield H., Garter C.A., Rodriguez P., Qiang Y.W., Vary C.P, & Reagan M.R. (2024). Inhibition of Acyl-CoA Synthetase Long Chain Isozymes Decreases Multiple Myeloma Cell Proliferation and Causes Mitochondrial Dysfunction. bioRxiv.

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Cell Cycle Analysis via Flow Cytometry

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For cell cycle analysis, cells were fixed in 70% ethanol at 4°C o/n. After fixation, cells were washed with PBS, and the DNA was stained with propidium iodide (PI). Samples were analyzed by Macsquant Analyzer (Miltenyi) and Flowlogic software. G2 checkpoint analysis was performed as described above, but cells were stained with antibodies against MPM2 or pHH3 to determine the number of mitotic cells. At least 15,000 cells were analyzed per condition, and three independent experiments were performed using a FACS Calibur (BD Biosciences) or a Macsquant Analyzer (Miltenyi) and analyzed using CellQuest or Macsquantify software, respectively. The number of mitotic cells after IR was divided by the number of mitotic cells in untreated conditions resulting in the relative mitotic entry (RME). For each experiment, the RME was normalized to the siLuciferase control, which was set to 1.

Warmerdam D.O., Alonso‐de Vega I., Wiegant W.W., van den Broek B., Rother M.B., Wolthuis R.M., Freire R., van Attikum H., Medema R.H, & Smits V.A. (2019). PHF6 promotes non‐homologous end joining and G2 checkpoint recovery. EMBO Reports, 21(1), e48460.

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Flow Cytometric Analysis of Cells

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For flow cytometric analysis, single cell suspensions were stained with the indicated antibodies (see Materials and Methods and Tables S1, S2) according to the manufacturer's instructions and analyzed using the MACSQuant™ Analyzer (Miltenyi Biotec). Cells were stained at a density of 0.5–2 × 10⁵ cells per sample in 50 μL volume of PBS pH 7.2, 2 mM EDTA, and 0.5% BSA (PEB) buffer at 2–8°C for 10 min, followed by a washing step with PEB. Cell viability was assessed by Propidium Iodide (PI, 1 μg/mL, Miltenyi Biotec) prior to flow cytometric analysis. Analysis was performed using the MACSQuant Analyzer and data analysis using the MACSQuatify software (Miltenyi Biotec).

Agorku D.J., Langhammer A., Heider U., Wild S., Bosio A, & Hardt O. (2019). CD49b, CD87, and CD95 Are Markers for Activated Cancer-Associated Fibroblasts Whereas CD39 Marks Quiescent Normal Fibroblasts in Murine Tumor Models. Frontiers in Oncology, 9, 716.

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Measuring Mitochondrial Membrane Potential and ROS

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To measure mitochondrial membrane potential, 200,000 cells per well were plated in 6-well plates, allowed to attach overnight, and treated with MBZ 24 hours later. After drug treatment, cells were washed twice with phosphate-buffered saline (PBS), and the mitochondrial membrane potential was measured via the fluorescent dye, tetramethylrhodamine methyl ester (TMRM). Cells were incubated with 50 nM TMRM for 30 minutes in PBS at 37°C, trypsinized, and washed with fresh media. After resuspending in PBS, cells were analyzed by flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec) for TMRM fluorescence. The mean fluorescence intensity was recorded for all the experimental groups. Three biologically independent experiments were performed.
For measuring reactive oxygen species (ROS) after MBZ treatment, 5 μM per well of the CellROX Deep Red Reagent (ThermoFisher Scientific) was added to the growth media at the indicated time points, and the cells were incubated for 30 minutes at 37°C. After the cells were washed with PBS, the cells were detached, resuspended in PBS, and analyzed by flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec) for CellROX Deep Red fluorescence (Excitation 635 nm).

Zhang L., Bochkur Dratver M., Yazal T., Dong K., Nguyen A., Yu G., Dao A., Bochkur Dratver M., Duhachek-Muggy S., Bhat K., Alli C., Pajonk F, & Vlashi E. (2018). Mebendazole Potentiates Radiation Therapy in Triple-Negative Breast Cancer. International journal of radiation oncology, biology, physics, 103(1), 195-207.

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Cell Proliferation Assay with NSC12

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Cells were seeded (5 × 10³) in 48-well cell culture plates in complete medium. At 24, 48, and 72 h, cells were detached and absolute cell counts were obtained by using the MACSQuant Analyzer (Miltenyi Biotec) and normalized in respect to time 0. Human HT-1080 fibrosarcoma cells were seeded (5 × 10³) in 48-well cell culture plates in complete medium, starved in 1% FBS for 24 h, and treated with DMSO or different concentration of NSC12 (0,1- 1- 3- 6- 10- 20 μM). At 24 or 48 h cells were detached, counted after propidium iodide labeling using the MACSQuant Analyzer (Miltenyi Biotec) and normalized in respect to DMSO-treated cells.

Rodrigues P.F., Matarazzo S., Maccarinelli F., Foglio E., Giacomini A., Silva Nunes J.P., Presta M., Dias A.A, & Ronca R. (2018). Long Pentraxin 3-Mediated Fibroblast Growth Factor Trapping Impairs Fibrosarcoma Growth. Frontiers in Oncology, 8, 472.

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Quantifying HER2 Expression and T-DM1 Binding

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To assess HER2 expression levels, cells were incubated either with phycoerythrin (PE)-labeled anti-human HER2 (24D2) or with corresponding isotype-control antibodies (BioLegend, San Diego, CA, USA) in buffer (1 % FBS, 2 mM EDTA and 0.1 % NaN₃ in PBS) for 30 min at 4 °C, after which they were washed and then analyzed using a MACSQuant Analyzer (Miltenyi Biotech K.K., Bergisch Gladbach, Germany). Before using the analyzer, 4 μg/ml propidium iodide (PI) (Sigma-Aldrich, St. Louis, MO, USA) was added to each sample to exclude dead cells.
To assess T-DM1 binding to PDA cells, 0.5x10⁶ cells were incubated with 30 μg/ml T-DM1 at 37 °C for 1 h. The cells were washed, incubated with PE-labeled anti-human IgG Fc (HP6017) (Biolegend) or corresponding isotype-control antibodies (Affymetrix, Santa Clara, CA, USA) in buffer for 30 min at 4 °C, washed, re-suspended and analyzed using a MACSQuant Analyzer (Miltenyi Biotech K.K.). Before using the analyzer, 4 μg/ml PI (Sigma-Aldrich) was added to the sample to exclude dead cells. The mean fluorescence intensity (MFI) of HER2 was analyzed using MACSQuantify Software.

Kan S., Koido S., Okamoto M., Hayashi K., Ito M., Kamata Y., Komita H., Nagasaki E, & Homma S. (2015). Up-regulation of HER2 by gemcitabine enhances the antitumor effect of combined gemcitabine and trastuzumab emtansine treatment on pancreatic ductal adenocarcinoma cells. BMC Cancer, 15, 726.

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Multiparametric Flow Cytometry Analysis

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Singe cell preparations were isolated from indicated tissue sites and flow stained. Fluorochrome-conjugated mAbs specific to mouse CD4 (clone GK1.5), CD19 (clone 6D5), CD25 (clone PC61.5), CD29 (clone HMβ1-1), CD39 (clone 24DMS1), CD40L (clone MR1), CD44 (clone IM7), CD49d (clone R1-2), CD62L (clone MEL-14), CD69 (clone H1.2F3), CD73 (Clone eBioTY/11.8), CD103 (clone 2E7), CD127 (clone A7R34), 4-1BB (clone 17B5), GITR (clone DTA-1), ICOS (clone 7E.17G9), LAG-3 (clone eBioC9B7W), LAP (clone TW7-16B4), PD-1 (clone 29F.1A12) and Thy1.1 (clone OX-7) (1:400 dilution, Biolegend or eBioscience) were used in various combinations for cell surface staining. Biotin-labeled anti-VISTA mAb was provided by Dr. Li Wang (Medical College of Wisconsin). The Fc receptor was blocked by CD16/32 antibody (Biolegend). For Foxp3 (1:400 dilution; clone FJK-16s, eBioscience) and CTLA-4 (1:400 dilution; clone UC10-4B9, Biolegend), cells were treated by eBioscience intracellular fixation/permeabilization buffer set and intracellular staining was performed. Stained cells were assayed by MACSQuant Analyzers (Miltenyi, Bergisch Gladbach, Germany).

Wang Y., Telesford K.M., Ochoa-Repáraz J., Haque-Begum S., Christy M., Kasper E.J., Wang L., Wu Y., Robson S.C., Kasper D.L, & Kasper L.H. (2014). An intestinal commensal symbiosis factor controls neuroinflammation via TLR2-mediated CD39 signaling. Nature communications, 5, 4432.

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