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Attune flow cytometer

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The Attune flow cytometer is a laboratory instrument used for analyzing and sorting cells or other particles in a fluid sample. It utilizes laser-based technology to detect and measure various physical and chemical characteristics of individual cells or particles as they pass through the instrument. The Attune flow cytometer is capable of providing quantitative data on the size, granularity, and fluorescence properties of the analyzed samples.

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Lab products found in correlation

Clear tissue culture plates, by Greiner (1 mentions) Kaluza, by Beckman Coulter (1 mentions) Universal blocking reagent, by BioGenex (1 mentions) Anti cd19 fitc, by BD (1 mentions) Anti cd11b pe, by BD (1 mentions) Anti cd69 pe, by BD (1 mentions) Mouse serum, by Merck Group (1 mentions) Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (1 mentions) Accutase, by Thermo Fisher Scientific (1 mentions) Sytox aadvanced dead cell stain, by Thermo Fisher Scientific (1 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions) Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions) Jetoptimus, by Polyplus Transfection (1 mentions)

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Attune NxT Flow Cytometer (19 citations) FACS Attune Flow Cytometer (2 citations)

9 protocols using attune flow cytometer

1

FRET Analysis of Cellular Processes

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All flow cytometry experiments were performed on a BD Attune flow cytometer (BD; Walthman, MA) equipped with violet (405nm) and blue (488nm) excitation lasers. mCitrine (530/30) and sensitized emission FRET (530/30) emission were excited with a 488nm and 405nm laser respectively. The FRET ratio was calculated on a cell by cell basis by programming a custom parameter (FRET/mCitrine ratio) using the BD attune cytometry software. Data was analyzed using FlowJo (FlowJo LLC, Ashland, OR) flow cytometry analysis software. To ensure accurate FRET measurements, dead cells and doublets were excluded from analysis using the forward and side scattering characteristics of the cells. With dead cells and doublets removed, cells were then gated based on their fluorescent intensity and only cells that were significantly brighter then non-transfected controls were used for analysis.
Voyton C.M., Qiu Y., Morris M.T., Ackroyd P.C., Suryadi J., Crowe L., Morris J.C, & Christensen K.A. (2018). A FRET flow cytometry method for monitoring cytosolic and glycosomal glucose in living kinetoplastid parasites. PLoS Neglected Tropical Diseases, 12(5), e0006523.
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2

BMDM Activation Marker Analysis

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BMDMs were seeded into 96-well µClear tissue culture plates (Greiner Bio-One) at 1 × 105 BMDMs per well and treated with varying concentrations of LCL161 or DMSO for 20 h. After washing cells with FACS PBS twice, cells were blocked with FcX block (1/100) for 5 min at 4 °C and were stained for the following cell surface markers: PE-conjugated CD40L (clone MR1, BioLegend), FITC-conjugated CD80 (Clone 16-10A1, BD Biosciences), AF647-conjugated anti-MHC I (H-2Kb, clone AF6-88.5, BD Pharmingen), and APC-eFluor 780-conjugated anti-MHC II (I-A/I-E, clone M5/114.15.2, eBiosciences) for 30 min at 4 °C in the dark. After cells were washed with FACS PBS twice, the expression level of activation markers on BMDMs was measured using BD Attune flow cytometer. Analysis was done using Kaluza (Beckman Coulter).
Kim D.S., Dastidar H., Zhang C., Zemp F.J., Lau K., Ernst M., Rakic A., Sikdar S., Rajwani J., Naumenko V., Balce D.R., Ewanchuk B.W., Tailor P., Yates R.M., Jenne C., Gafuik C, & Mahoney D.J. (2017). Smac mimetics and oncolytic viruses synergize in driving anticancer T-cell responses through complementary mechanisms. Nature Communications, 8, 344.
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3

FcεRI and TLR-4 Expression in BMMCs

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For each condition, 106 WT or R6/1 BMMCs were incubated with IgE (100 ng/mL) for 24 h. Then, the cells were collected and re-suspended in universal blocking reagent (BioGenex, San Ramon, CA) for 5 min at 4 °C. The cells were washed twice with 1× PBS and incubated with anti-FcεRI (1:500 in blocking buffer containing 1× PBS, 5 g/L BSA, 0.5 g/L sodium azide), and anti-TLR-4 N-terminal Ab (1:100 in blocking buffer) for 30 min at 4 °C. The cells were washed and incubated with a PE-coupled anti-IgG Ab (1:200 in blocking buffer). After 30 min of incubation at 4 °C, the cells were washed again and re-suspended in 1% paraformaldehyde (PFA). An anti-isotype staining was performed at the same time. The samples were analyzed in a CytoFLEX LX (B-R-V-Y-N-I) flow cytometer (Beckman Coulter, Brea, CA, USA), using the software CytExpert for data acquisition; plots showing PE+ populations were generated with the software Kaluza Analysis. An Attune flow cytometer (Beckton Dickinson, Franklin Lakes, NJ, USA) was used in some experiments.
Pérez-Rodríguez M.J., Ibarra-Sánchez A., Román-Figueroa A., Pérez-Severiano F, & González-Espinosa C. (2020). Mutant Huntingtin affects toll-like receptor 4 intracellular trafficking and cytokine production in mast cells. Journal of Neuroinflammation, 17, 95.
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4

Flow Cytometric Immunophenotyping of CLL Cells

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Cell suspensions from PB, LN and BM were washed and blocked with 10% mouse serum (Sigma). Samples were then stained with anti-CD19-FITC (Becton Dickinson), PI, and either anti-CD69-PE or anti-CD11b-PE (Becton Dickinson) and analyzed in an Attune flow cytometer. In each quantification experiment, a constant calibrator sample was stained simultaneously. Expression data was reported as the percentage of viable CLL cells positive for CD69 or CD11b after subtraction of cells stained for IgG1 isotype-PE (Becton Dickinson).
Montraveta A., Lee-Vergés E., Roldán J., Jiménez L., Cabezas S., Clot G., Pinyol M., Xargay-Torrent S., Rosich L., Arimany-Nardí C., Aymerich M., Villamor N., López-Guillermo A., Pérez-Galán P., Roué G., Pastor-Anglada M., Campo E., López-Guerra M, & Colomer D. (2015). CD69 expression potentially predicts response to bendamustine and its modulation by ibrutinib or idelalisib enhances cytotoxic effect in chronic lymphocytic leukemia. Oncotarget, 7(5), 5507-5520.
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5

Apoptosis and Cell Cycle Analysis

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A549 cells were treated with C188-9 or PL (@30μM) for up to 48 hrs. Cells were dissociated into singlets followed by staining with FITC-conjugated antibodies for Annexin V analysis (Becton Dickinson, catalog #556547) or propidium iodide stain solution for cell cycle analysis (Becton Dickinson, catalog #340242). Cells were analyzed on an Attune flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Lewis K.M., Bharadwaj U., Eckols T.K., Kolosov M., Kasembeli M.M., Fridley C., Siller R, & Tweardy D.J. (2015). SMALL-MOLECULE TARGETING OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION (STAT) 3 TO TREAT NON-SMALL CELL LUNG CANCER. Lung cancer (Amsterdam, Netherlands), 90(2), 182-190.
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6

Quantifying Cell Surface Glycans

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Cells (2 × 105/well) were collected and washed with cold phosphate-buffered saline (PBS) on ice. Cells were incubated with or without biotinylated lectins (E4-PHA) in 0.1% BSA/PBS for 30 min and then stained with Alexa Fluor 647 for 25 min on ice. Then, cells were washed three times and resuspended in 1 ml of 0.1% BSA/PBS and analyzed using Attune flow cytometer (BD Biosciences).
Song W., Liang C., Sun Y., Morii S., Yomogida S., Isaji T., Fukuda T., Hang Q., Hara A., Nakano M, & Gu J. (2023). Expression of GnT-III decreases chemoresistance via negatively regulating P-glycoprotein expression: Involvement of the TNFR2-NF-κB signaling pathway. The Journal of Biological Chemistry, 299(4), 103051.
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7

Apoptosis Assay of Retinal Ganglion Cells

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RGCs with small parts of optic vesicles (OVs) were removed from the whole cell body, picked from the plate, added to 100 µL of PBS, and agitated; these were then centrifuged at 300× g for 3 min. After aspirating the supernatant, accutase (Gibco) was added to the cell precipitates and kept at 37 °C for 5 min. After pipetting 10 times, CellEventTM Caspase-3/7 Green Detection Reagent (0.5 M; Invitrogen) was added, agitated, and kept at 37 °C for 25 min in darkness. SYTOXTM AADvancedTM Dead Cell Stain (1M; Invitrogen) was added, agitated, and incubated at 37 °C for 10 min in darkness. Next, 400 µL of PBS was added and filtered through a round-bottom tube with a 35 µm strainer cap.
An Attune flow cytometer (BD BioSciences, San Jose, CA, USA) was used for the flow cytometry. The analysis was conducted five times with a minimum of 20,000 cells per condition.
Yoshida T., Yokoi T., Tanaka T., Matsuzaka E., Saida Y., Nishina S., Takada S., Shimizu S, & Azuma N. (2024). Modeling of Retina and Optic Nerve Ischemia–Reperfusion Injury through Hypoxia–Reoxygenation in Human Induced Pluripotent Stem Cell-Derived Retinal Ganglion Cells. Cells, 13(2), 130.
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8

Lectin-Mediated Cell Glycosylation Analysis

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Cells were detached by brief exposure to 0.25% trypsin containing 1 mM EDTA and resuspended at 1 × 106 cells/ml density. After washing with ice-cold PBS, equal amounts of cells were incubated with biotinylated lectins (SNA or MAA) in 0.1% BSA in PBS for 30 min on ice. Subsequently, the cells were incubated with streptavidin conjugate Alexa Fluor 647 (1:500) for 25 min at RT in the dark. Then, the cells were washed and resuspended in 1 ml MACS buffer (PBS containing 0.1% BSA). The fluorescence intensities were detected by Attune flow cytometer (BD Biosciences) and analyzed using FlowJo software.
Sun Y., Isaji T., Oyama Y., Xu X., Liu J., Hanamatsu H., Yokota I., Miura N., Furukawa J.I., Fukuda T, & Gu J. (2023). Focal-adhesion kinase regulates the sialylation of N-glycans via the PI4KIIα-PI4P pathway. The Journal of Biological Chemistry, 299(8), 105051.
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9

Quantifying Intracellular H2O2 Levels

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Chemical probe based: Intracellular H2O2 level was measured as in (Iwashita et al., 2021 (link)). Briefly, murine PDAT-sgRosa26 or PDAT-sgMsrA cells were plated on 6-well plates and cultured overnight. Cells were then washed with PBS and incubated with PG1-FM (5 μM) or vehicle (DMSO) in HBSS (Gibco Cat# 14025092) for 30 min. Cells were harvested by 0.05% Trypsin with EDTA (Gibco Cat# 25300054), filtered and analyzed by flow cytometry.
roGFP biosensor based: Subcellular H2O2 and GSH redox potential was monitored using roGFP2 biosensors. Briefly, cells were transiently transfected with roGFP2 using jetOPTIMUS (Polyplus Cat# 101000025). Redox status was evaluated using the Attune flow cytometer, with 405, 488 and 531 nm lasers (BD Biosciences, San Jose, CA) and the flow cytometry data was analyzed using the FlowJo software (BD Biosciences). Voltage settings for the SSC, FSC, 405 nm and GFP channels were kept constant for all experiments. Measurements were taken at 405 and 488 nm for calculations of redox ratio. Each analyzed population had a sample size of 10,000 transfected cells.
He D., Feng H., Sundberg B., Yang J., Powers J., Christian A.H., Wilkinson J.E., Monnin C., Avizonis D., Thomas C.J., Friedman R.A., Kluger M.D., Hollingsworth M.A., Grandgenett P.M., Klute K.A., Toste F.D., Chang C.J, & Chio I.I. (2022). Methionine oxidation activates pyruvate kinase M2 to promote pancreatic cancer metastasis. Molecular cell, 82(16), 3045-3060.e11.
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