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Macsquant10 analyzer

Manufactured by Miltenyi Biotec
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Sourced in Germany, United States

The MACSQuant10 Analyzer is a compact flow cytometry instrument designed for automated high-throughput analysis of cell samples. It features a high-performance optical system, multiple fluorescence detection channels, and intuitive software for data acquisition and analysis.

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Lab products found in correlation

Rbc lysis buffer, by Thermo Fisher Scientific (3 mentions) Bovine serum albumin (bsa), by Miltenyi Biotec (2 mentions) 7 aad staining, by BD (2 mentions) Anti fc af647 conjugate, by Jackson ImmunoResearch (2 mentions) Macsquantify software, by Miltenyi Biotec (2 mentions) Sytox red, by Thermo Fisher Scientific (1 mentions) Accutase, by STEMCELL (1 mentions) Celltrace violet, by Thermo Fisher Scientific (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Tc20 automated cell counter, by Bio-Rad (1 mentions) Cd8 pe, by Miltenyi Biotec (1 mentions) Viobility 405 520, by Miltenyi Biotec (1 mentions) Inside stain, by Miltenyi Biotec (1 mentions) Anti ifnγ apc, by Miltenyi Biotec (1 mentions) Cd3 vioblue, by Miltenyi Biotec (1 mentions) Cd4 fitc, by Miltenyi Biotec (1 mentions) Ficoll paque, by GE Healthcare (1 mentions) Propidium iodide, by Thermo Fisher Scientific (1 mentions) Rnase a, by Thermo Fisher Scientific (1 mentions) Annexin 5 fitc pi apoptosis kit, by MultiSciences Biotech (1 mentions)

34 protocols using macsquant10 analyzer

1

CAR T-Cell Surface Expression Quantification

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For cell staining, half a million CAR T-transduced cells were harvested from culture, washed two times in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec), and CAR surface expression detected by staining with CD19-Fc peptide (R&D, Minneapolis, MN) followed by anti-Fc-AF647 conjugate (Jackson Immuno Research, West Grove, PA). Non-transduced cells were used as negative controls. Dead cells in all studies were excluded by 7AAD staining (BD Biosciences, San Jose, CA, USA). Cells were washed twice and resuspended in 200 μL Staining Buffer before quantitative analysis by flow cytometry. Flow cytometric analysis was performed on a MACSQuant®10 Analyzer (Miltenyi Biotec), and data plots were generated using FlowJo software (Ashland, OR, USA).
Maschan M., Caimi P.F., Reese-Koc J., Sanchez G.P., Sharma A.A., Molostova O., Shelikhova L., Pershin D., Stepanov A., Muzalevskii Y., Suzart V.G., Otegbeye F., Wald D., Xiong Y., Wu D., Knight A., Oparaocha I., Ferencz B., Roy A., Worden A., Kruger W., Kadan M., Schneider D., Orentas R., Sekaly R.P., de Lima M, & Dropulić B. (2021). Multiple site place-of-care manufactured anti-CD19 CAR-T cells induce high remission rates in B-cell malignancy patients. Nature Communications, 12, 7200.
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2

Intracellular ROS Measurement by Flow Cytometry

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To measure the level of intracellular ROS, cells were incubated with 500nM CellRox Green in media for 1hour. Following incubation cells were washed twice with PBS and then detached from plates by Accutase (StemCell Technologies) treatment for 15 minutes. Cells were then spun down (1000x g, 3 min) and resuspended in 2% FBS in PBS and stained with 5nM SyTOX Red (ThermoFisher) to measure cell viability. The samples were analyzed on a MACs Quant 10 Analyzer (Miltenyi Biotec) at the University of Wisconsin Carbone Cancer Center Flow Cytometry Laboratory. Resulting data files were analyzed using FlowJo10.4 (Tree Star). Median CellRox Green signal of those cells that were SyTOX Red negative (live cells) was calculated. See example of gating strategy in Supplementary Figure 11b.
Seim G.L., Britt E.C., John S.V., Yeo F.J., Johnson A.R., Eisenstein R.S., Pagliarini D.J, & Fan J. (2019). Two-stage metabolic remodelling in macrophages in response to lipopolysaccharide and interferon-γ stimulation. Nature metabolism, 1(7), 731-742.
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3

High-Throughput Phenotyping of Engineered NK Cells

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For all experiments, flowcytometry was carried out using a MACSQuant Analyzer 10. Propidium iodide staining was employed to identify viable cells. Antibodies were from Miltenyi Biotec and included in addition to the human MACS marker screen monoclonal antibodies targeting CD3, CD19, CD56, and CX3CR1. CD19-CAR expression was detected by using a recombinant human CD19 Fc (R&D Systems) fusion protein at a concentration of 25 μg/mL followed by incubation with a monoclonal anti-Fc antibody conjugated to APC (Miltenyi Biotec). Of note, to remove nonspecific background staining from NK cells was minimized by incubation of the cells for 48 h in serum free culture medium prior to the staining. Where indicated, proliferation rates were determined by flow cytometry by labeling of the cells with CellTrace Violet (Thermo Fisher) at a concentration of 10 μM. High-throughput surface marker screening was carried out 3–4 days after transduction and CellTrace Violet labeled NK cells were incubated with the MACS Marker Screen human antibody panel targeting 371 surface markers. Detection was carried out by automated flowcytometry using a MACSQuant 10 Analyzer (Miltenyi Biotech).
Bari R., Granzin M., Tsang K.S., Roy A., Krueger W., Orentas R., Schneider D., Pfeifer R., Moeker N., Verhoeyen E., Dropulic B, & Leung W. (2019). A Distinct Subset of Highly Proliferative and Lentiviral Vector (LV)-Transducible NK Cells Define a Readily Engineered Subset for Adoptive Cellular Therapy. Frontiers in Immunology, 10, 2001.
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4

OX40 and OX40L Expression Profiling

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OX40 and OX40L expression was measured by CHDR (Leiden, The Netherlands) on cell subsets of whole blood samples using flow cytometry. Red blood cell lysis was performed on heparinized whole blood using RBC lysis buffer (Thermo Fisher, Waltham, MA, USA). Leukocytes were stained with fluorochrome labelled antibodies at 4°C for 30 minutes, see Table S1 for a complete list. After staining, the cells were washed with PBS (Thermo Fisher). Samples were measured on a MACSQuant 10 analyzer, and analyzed using MACSQuantify software (both Miltenyi Biotec, Bergisch‐Gladbach, Germany). See Figure S2 for the gating strategy. OX40 expression was assessed in CD4+ and CD8+ T cells, regulatory T cells and Th17 cells and OX40L expression was assessed in CD19+ and CD14+ monocytes in all cohorts. In addition, expression of OX40 and OX40L in CD4+ and CD8+ effector memory and central memory cells was assessed in cohorts 4–8.
Saghari M., Gal P., Gilbert S., Yateman M., Porter‐Brown B., Brennan N., Quaratino S., Wilson R., Grievink H.W., Klaassen E.S., Bergmann K.R., Burggraaf J., van Doorn M.B., Powell J., Moerland M, & Rissmann R. (2022). OX40L Inhibition Suppresses KLH‐driven Immune Responses in Healthy Volunteers: A Randomized Controlled Trial Demonstrating Proof‐of‐Pharmacology for KY1005. Clinical Pharmacology and Therapeutics, 111(5), 1121-1132.
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5

F-TIL Viability, Identity, and Potency Assays

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The acceptance criteria for F-TIL included a viability assay, trypan blue exclusion, measured via TC20 Automated Cell Counter (BioRad). The acceptance is set at not less than (NLT) 70% viable. Also, cell density and cell count were determined from this measurement with an accepted cell count of NLT 1×109 cells. The F-TIL identity and potency were established from an interferon gamma (IFNɣ) release assay. IFNɣ+ T cells were assayed by intracellular staining with and without soluble CD3 (OKT3, Miltenyi Biotec) stimulation. Briefly, after control or OKT3 stimulation with Golgi Plug and Golgi Stop (Miltenyi Biotec), cells were pelleted and stained with Viobility 405/520 (Miltenyi Biotec), followed by CD3-VioBlue, CD4-FITC, and CD8-PE (all from Miltenyi Biotec). Lastly, cells were treated with Inside Stain (Miltenyi Biotec) and then stained with anti-IFNγ-APC (Miltenyi Biotec). Cellular preparations were assayed using a Miltenyi MACSQuant 10 analyzer.
Ahrens E.T., Helfer B.M., O’Hanlon C.F., Lister D.R., Bykowski J.L., Messer K., Leach B.I., Chen J., Xu H., Daniels G.A, & Cohen E.E. (2023). Method for estimation of apoptotic cell fraction of cytotherapy using in vivo fluorine-19 magnetic resonance: pilot study in a patient with head and neck carcinoma receiving tumor-infiltrating lymphocytes labeled with perfluorocarbon nanoemulsion. Journal for Immunotherapy of Cancer, 11(6), e007015.
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6

Isolation of PBMCs from Buffy Coats

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Buffy coats were diluted [1:3] with PBS and added to Leukosep tubes containing Ficoll-Paque (GE Healthcare). Tubes were centrifuged at 450g for 40 min and the resulting buffy layer extracted. Isolated PBMCs were then washed in PBS and counted on MACSQuant10 Analyzer (Miltenyi Biotec).
Cooper R.S., Fraser A.R., Smith L., Burgoyne P., Imlach S.N., Jarvis L.M., Turner D.M., Zahra S., Turner M.L, & Campbell J.D. (2021). Rapid GMP-Compliant Expansion of SARS-CoV-2–Specific T Cells From Convalescent Donors for Use as an Allogeneic Cell Therapy for COVID-19. Frontiers in Immunology, 11, 598402.
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7

Cell Cycle Analysis by Flow Cytometry

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Flow cytometric cell-cycle analysis was performed as described [48 (link)], with minor modifications. Briefly, untreated and Pg-treated cells were fixed, treated with Rnase A (12.5 µg/mL) (Thermo-Fisher Scientific, Milan, Italy), stained with propidium iodide (40 µg/mL) (Invitrogen, Thermo-Fisher Scientific, Milan, Italy), and analyzed by flow cytometry using a MACSQuant10 Analyzer (Miltenyi Biotec GmbH, Bielefeld, Germany) for cell-cycle status. Data were analyzed using FlowJo v10.6.2 software (TreeStar, Ashland, OR, USA).
Tamburello M., Abate A., Rossini E., Basnet R.M., Zizioli D., Cosentini D., Hantel C., Laganà M., Tiberio G.A., Grisanti S., Memo M., Berruti A, & Sigala S. (2023). Preclinical Evidence of Progesterone as a New Pharmacological Strategy in Human Adrenocortical Carcinoma Cell Lines. International Journal of Molecular Sciences, 24(7), 6829.
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8

CAR T-cell Surface Expression Analysis

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For cell staining, half a million CAR T-transduced cells were harvested from culture, washed two times in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec), and CAR surface expression detected by staining with CD33-Fc peptide (R&D, Minneapolis, MN) followed by anti Fc-AF647 conjugate (Jackson ImmunoResearch, West Grove, PA). Non-transduced cells were used as negative controls. Dead cells in all studies were excluded by 7AAD staining (BD Biosciences, San Jose, CA). Cells were washed twice and resuspended in 200 ul Staining Buffer before quantitative analysis by flow cytometry. Flow cytometric analysis was performed on a MACSQuant® 10 Analyzer (Miltenyi Biotec), and data plots were generated using FlowJo software (Ashland, OR).
Schneider D., Xiong Y., Hu P., Wu D., Chen W., Ying T., Zhu Z., Dimitrov D.S., Dropulic B, & Orentas R.J. (2018). A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia. Frontiers in Oncology, 8, 539.
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9

Cell Viability and Apoptosis Evaluation

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Cell viability was evaluated via a Cell Counting Kit-8 based on the manufacturer’s protocols; Absorbance measurements at 450 nm were taken an EXL800 microplate reader (BioTek Instruments). For the analysis of cell apoptosis, cells were stained with an Annexin V-FITC/PI apoptosis kit (MULTI SCIENCES, China). Annexin V-positive cells were assessed using a MACSQuant 10 Analyzer (Miltenyi Biotec), and the data were processed with FlowJo software (version 7.6.1; BD Company).
Peng G., Wang C., Wang H., Qu M., Dong K., Yu Y., Jiang Y., Gan S, & Gao X. (2023). Gankyrin-mediated interaction between cancer cells and tumor-associated macrophages facilitates prostate cancer progression and androgen deprivation therapy resistance. Oncoimmunology, 12(1), 2173422.
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10

Analyzing NOK Cell Viability and Apoptosis

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Flow cytometry analysis of NOK cells was used to assay for cell viability and mechanism of cell death. NOKs were grown to semi confluence (1.5 x 104 cells per well) in 96-well plates (0.33 cm2 area) (Nunc, Rochester, NY), and treated with PrtFAC (10, 50, and 100 μM) for 48 h. Post treatment, adherent cells were trypsinized and washed with 1X PBS and centrifuged (at 500 g for 5 min) in sterile FACS 5 ml centrifuge tubes. The supernatant was discarded and the cells were stained for flow cytometry analysis using pacific blue™ Annexin-V and 7-Aminoactinomycin D (7-AAD) [Biolegend, San Diego, CA], according to the manufacturer’s instructions. Test volumes were decreased for staining in a 96 well plate. Briefly, cells were resuspended in 10 μl of Biolegend Annexin V binding buffer then stained with 0.5 μl of pacific blue™ Annexin V and 1 μl of 7-AAD for 15 min at room temperature in the dark. Cells were washed with 200 μl of Annexin-V binding buffer then resuspended in 1% paraformaldehyde. TRAIL protein (20 μM) was used as a positive control, while Z-VAD-FMK (20 μM), a pan-caspase inhibitor, was used as a caspase control. Data acquisition and analysis of apoptotic cells were performed using the MACSQuant 10 Analyzer (MiltenyiBiotec, Inc, Auburn CA) and FlowJo vX Data Analysis Software (FlowJo, Ashland, OR).
Chioma O., Aruni A.W., Milford T.A, & Fletcher H.M. (2016). Filifactor alocis collagenase can modulate apoptosis of normal oral keratinocytes. Molecular oral microbiology, 32(2), 166-177.
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