Attune nxt flow cytometer

Manufactured by Tree Star

The Attune NxT flow cytometer is a versatile instrument used for the analysis and sorting of cells and other particles. It employs the principle of flow cytometry to rapidly analyze and categorize individual cells within a sample. The Attune NxT measures various parameters, including size, granularity, and fluorescence intensity, providing researchers with detailed information about the composition and characteristics of the analyzed population.

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Attune NxT (3 citations)

12 protocols using attune nxt flow cytometer

Splenocyte, Lymph Node, and Brain Cell Isolation

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Preparation of single-cell suspensions from spleens, LNs and brains is described above. Nonspecific binding was blocked using an Fc receptor-blocking solution (TruStain FcX, BioLegend, 101320, 1:200) for 10 min at 4 °C before immunostaining. Subsequently, the cells were stained with corresponding antibodies for 30 min at 4 °C. Then, cells were washed to remove excess antibodies and resuspended in FACS buffer. Samples were run on an Attune NxT flow cytometer and then analysed using FlowJo software (10.8.1, Tree Star).

Yin X., Zhang S., Lee J.H., Dong H., Mourgkos G., Terwilliger G., Kraus A., Geraldo L.H., Poulet M., Fischer S., Zhou T., Mohammed F.S., Zhou J., Wang Y., Malloy S., Rohner N., Sharma L., Salinas I., Eichmann A., Thomas J.L., Saltzman W.M., Huttner A., Zeiss C., Ring A., Iwasaki A, & Song E. (2024). Compartmentalized ocular lymphatic system mediates eye–brain immunity. Nature, 628(8006), 204-211.

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SARS-CoV-2 S Protein Antibody Detection

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293 T cells were transfected with a plasmid encoding the full-length S protein (kindly provided by O. Schwartz) [39 ] or a control plasmid using Lipofectamine 2000 (Life technologies). Twenty-four hours post-transfection, the cells were detached using PBS-EDTA and transferred into U-bottom 96-well culture plates (200.000 cells/well). Cells were saturated with 10% fetal bovine serum at 4 C for 10 min and incubated with the patients’ sera (1:300 dilution) in PBS containing 0.5% BSA for 30 minutes at 4 °C. Cells were then washed and stained for 30 min at 4 °C using a goat anti-Human IgG (Fc specific)-FITC antibody that specifically recognizes the Fc fragment and not the light chains of the Ig (Sigma). After washing, cells were fixed with 2% PFA and analyzed on an Attune™ Nxt flow cytometer using FlowJo software (Tree Star, Inc.).

André S., Azarias da Silva M., Picard M., Alleaume-Buteau A., Kundura L., Cezar R., Soudaramourty C., André S.C., Mendes-Frias A., Carvalho A., Capela C., Pedrosa J., Gil Castro A., Loubet P., Sotto A., Muller L., Lefrant J.Y., Roger C., Claret P.G., Duvnjak S., Tran T.A., Zghidi-Abouzid O., Nioche P., Silvestre R., Corbeau P., Mammano F, & Estaquier J. (2022). Low quantity and quality of anti-spike humoral response is linked to CD4 T-cell apoptosis in COVID-19 patients. Cell Death & Disease, 13(8), 741.

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Mitochondrial Function Assessment in BMDMs

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BMDMs were seeded in non-tissue-culture plates and stimulated as described above. Cells were stained with MitoTracker Green (for total mitochondrial mass) and MitoTracker Red CMXRos (for mitochondrial membrane potential) or MitoSOX (for mitochondrial ROS) according to manufacturer’s instructions (Invitrogen) in combination with Live/Dead stain (eBioscience™ Fixable Viability Dye eFluor™ 780). For flow cytometry analysis, data (10,000 events per sample) was acquired with the Attune NxT Flow Cytometer and analysed with FlowJo v10 software (TreeStar). A flow gating strategy is provided in Fig. 5 of Supplementary Information.

Dowling J.K., Afzal R., Gearing L.J., Cervantes-Silva M.P., Annett S., Davis G.M., De Santi C., Assmann N., Dettmer K., Gough D.J., Bantug G.R., Hamid F.I., Nally F.K., Duffy C.P., Gorman A.L., Liddicoat A.M., Lavelle E.C., Hess C., Oefner P.J., Finlay D.K., Davey G.P., Robson T., Curtis A.M., Hertzog P.J., Williams B.R, & McCoy C.E. (2021). Mitochondrial arginase-2 is essential for IL-10 metabolic reprogramming of inflammatory macrophages. Nature Communications, 12, 1460.

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In vitro synNotch-CAR T cell Assay

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For in vitro synNotch-CAR T cell and tumor cell coculture experiments, 1 × 10⁴ tumor cells (either U87 or GBM6) were cultured overnight in a flat-bottom 96-well tissue culture plate. The next morning, 1 × 10⁴ to 5 × 10⁴ T cells were added to the plate and cocultures were analyzed for 24 to 96 hours for activation and specific lysis of tumor cells. For in vitro synNotch T cell stimulations cocultured with three different cell populations, an additional 1 × 10⁴ priming cells (either K562 or L929) were added to the initial overnight culture. Flow cytometry was performed using BD LSRII or Attune NxT Flow Cytometer; analysis was performed by FlowJo software (Tree Star).

Choe J.H., Watchmaker P.B., Simic M.S., Gilbert R.D., Li A.W., Krasnow N.A., Downey K.M., Yu W., Carrera D.A., Celli A., Cho J., Briones J.D., Duecker J.M., Goretsky Y.E., Dannenfelser R., Cardarelli L., Troyanskaya O., Sidhu S.S., Roybal K.T., Okada H, & Lim W.A. (2021). SynNotch-CAR T cells overcome challenges of specificity, heterogeneity, and persistence in treating glioblastoma. Science translational medicine, 13(591), eabe7378.

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Isolation and Phenotyping of Brain-Infiltrating T Cells

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For all experiments involving phenotyping of adoptively transferred engineered T cells, brain and spleen were harvested after per-fusion with cold PBS. Brains were mechanically minced and treated at 37°C for 30 min with digestion mix consisting of collagenase D (30 mg/ml) and deoxyribonuclease (10 mg/ml) and soybean trypsin inhibitor (20 mg/ml). The resulting brain homogenate was resuspended in 70% Percoll (GE Healthcare), overlaid with 30% Percoll, and then centrifuged for 30 min at 650g. Enriched brain infiltrating T cells were recovered at the 70–30% interface and stained with fluorescently conjugated antibodies against CD3 (5 µl; catalog no. 555342, BD Biosciences) and CD45 (5 µl; catalog no. 564357, BD Biosciences) for 1 hour at 4°C. Before staining with antibodies, cells were stained with BD Horizon Fixability Viability Stain 780 (BD Biosciences) to discriminate live from dead cells. Data were collected on the Attune NxT Flow Cytometer, and the analysis was performed in FlowJo software (Tree Star).

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Isolation and Quantification of Spheroid Cells

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Spheroids were collected from the microwells after 24 hours, washed with PBS without Ca²⁺, Mg²⁺, disintegrated into single cells by 15-minute incubation in TrypLE solution (Gibco) and through pipetting. Single cells were stained with Zombie Violet Fixable Viability Kit (BioLegend), washed with PBS and stained for 30 minutes with anti-human CD29 and CD49e antibodies (BioLegend). Sample acquisition was performed on Attune NxT flow cytometer and the data were analyzed with FlowJo software (TreeStar).

Zarubova J., Hasani-Sadrabadi M.M., Norris S.C., Majedi F.S., Xiao C., Kasko A.M, & Li S. (2022). Cell-Taxi: Mesenchymal Cells Carry and Transport Clusters of Cancer Cells. Small (Weinheim an der Bergstrasse, Germany), 18(50), e2203515.

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Cytokine Production and Apoptosis in CD4+ T Cells

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Where indicated, CD4⁺ cells were stimulated with PMA (50 ng/mL; Sigma) and Ionomycin (750 ng/mL; EMD Millipore) for 5 h in the presence of GolgiPlug (BD Biosciences). Intracellular staining was performed as previously described(86 (link)). LIVE/DEAD Fixable near-IR Dead Cell Stain (Invitrogen) was used to exclude dead cells. Staining of CD4⁺ T cells was performed with antibodies to CD4 (RM4–5), IL-6Rα (D7715A7), IL-17A (TC11–18H10), TCR-β (H57–597) and Foxp3 (FJK-16s) from BD Biosciences, IFN-γ (XMG1.2), TCR-β (H57–597), gp130 (KGP130), and Thy1.1 (His51) from eBioscience, and Thy1.1 (OX-7) from Biolegend. For apoptosis analysis, annexin V and propidium iodide staining was performed using an Annexin V Apoptosis Detectin Kit (eBioscience) according to manufacturer’s instructions. Samples were acquired on an LSRII or Attune NxT flow cytometer and data were analyzed with FlowJo software (Tree Star).

Harbour S.N., DiToro D.F., Witte S.J., Zindl C.L., Gao M., Schoeb T.R., Jones G.W., Jones S.A., Hatton R.D, & Weaver C.T. (2020). Th17 Cells Require Ongoing Classic IL-6 Receptor Signaling to Retain Transcriptional and Functional Identity. Science immunology, 5(49), eaaw2262.

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Lung Cell Fractionation and Analysis

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Lung cell suspensions were prepared by collagenase digestion of whole lung tissue (Miltenyi Biotec, Bergisch Gladbach, Germany) from healthy and infected animals. RBCs were lysed with lysis buffer (Thermo Fisher). Gating strategy was adopted from as described previous after exclusions of doublets and debris (Misharin et al., 2013 (link)). Briefly, the following expression patterns were used to identify alveolar macrophages (Siglec F⁺ CD11b⁻CD11c⁺ CD64⁺), CD103 ⁺ dendritic cells (CD11C⁺ CD103⁺), neutrophils (CD11b⁺ Ly6G⁺), eosinophils (Siglec F⁺ CD11b⁺ CD11c⁻), Ly6C ⁺ monocytes (CD11b⁺ MHCII⁻ CD64^+/− Ly6C⁺) and CD11b⁺ dendritic cells (CD11b⁺ MHCII⁺ CD11c⁺ CD24⁻ CD64⁻). Cells were washed and re-suspended in Fluorescent Activated Cell Sorting (FACS) buffer. Data was collected on the Attune NXT flow cytometer and analyzed with FlowJo software (Tree Star, Inc., Ashland OR).

Kottom T.J., Nandakumar V., Hebrink D.M., Carmona E.M, & Limper A.H. (2020). A Critical Role for CARD9 in Pneumocystis Pneumonia Host Defense. Cellular microbiology, 22(10), e13235.

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Treg and Effector T Cell Profiling

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Method as described in Alwarawrah et al. 2020 [31 (link)]. Treg staining: Two million splenocytes were fixed and permeabilized using the Foxp3 Transcription Factor Staining Buffer kit (eBioscience) and stained for Foxp3 following the manufacturer instructions. For the identification of Treg cells, the following antibodies were used: BV421 Armenian Hamster anti-mouse CD3e (Biolegend, San Diego, CA), BV605 rat anti-mouse CD4 (Biolegend), PeCy7 rat anti-mouse CD25 (Biolegend), AF488 rat anti-mouse Foxp3 (Biolegend). Cytokine staining: For the identification of effector T cells (Th1 and Th17) and evaluation of their function, the following antibodies were used: BV421 Armenian Hamster anti-mouse CD3e (BD BioSciences), BV605 rat anti-mouse CD4 (Biolegend), AF488 rat anti-mouse CD8a (Biolegend), APC rat anti-mouse IL-17A (Biolegend), and PE/Cy7 rat anti-mouse IFNγ (Biolegend). Five million splenocytes were stimulated for 4.5 h in complete media containing Golgi Plug (2 μg/ml) (BD Biosciences), PMA (50 ng/ml) (Sigma-Aldrich, St. Louis, MO), and ionomycin (1 μg/ml) (Sigma-Aldrich), then permeabilized and fixed with Cytofix/Cytoperm kit (BD Biosciences) and stained for IFN-γ (Biolegend) and IL-17A (Biolegend) following the manufacturer’s protocol. Samples were acquired on a ThermoFisher Attune NxT flow cytometer, and data were analyzed using FlowJo (Treestar, Ashland, OR).

Kiernan K., Nichols A.G., Alwarawrah Y, & MacIver N.J. (2023). Effects of T cell leptin signaling on systemic glucose tolerance and T cell responses in obesity. PLOS ONE, 18(6), e0286470.

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ADCC Assay for Targeted Cell Killing

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Killing assay performed as previously described(59 (link)) with some modifications. Briefly, p815 cells (a mouse leukemia line) were stained with CellTrace Violet Cell Proliferation Kit (ThermoFisher) at 37°C for 20 mins. Reaction was stopped by addition of RPMI (Corning) with 10% FBS at 37C for 5 mins. Stained p815 cells were either opsonized with 10 μg/ml p815 rat anti-mouse antibody (Clone: 2.4G2, BD Biosciences) or left uncoated for 30 mins at 37°C. After incubation, PBMCs were cocultured with opsonized and uncoated target cells at an effector:target ratio of 10:1. Co-cultures were incubated at 37°C for 5 hours, before staining for dead and dying cells using 7-AAD Viability staining solution (1:25, Biolegend) and PE Annexin V (1:20, Biosciences) in 1x BD Annexin V Binding Buffer for 15 mins at room temperature. Volume was brought up to 200 μl and analyzed on the Attune NXT Flow Cytometer within the hour and analyzed with FlowJo X (Tree Star) software. Percentage of killed target cells was calculated by subtracting the percentages of dead uncoated target cells from the percentages of dead opsonized target cells.

Ty M., Sun S., Callaway P.C., Rek J., Press K.D., van der Ploeg K., Nideffer J., Hu Z., Klemm S., Greenleaf W., Donato M., Tukwasibwe S., Arinaitwe E., Nankya F., Musinguzi K., Andrew D., de la Parte L., Mori D.M., Lewis S.N., Takahashi S., Rodriguez-Barraquer I., Greenhouse B., Blish C., Utz P., Khatri P., Dorsey G., Kamya M., Boyle M., Feeney M., Ssewanyana I, & Jagannathan P. (2023). Malaria-driven expansion of adaptive-like functional CD56-negative NK cells correlates with clinical immunity to malaria. Science translational medicine, 15(680), eadd9012.

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