Accuri c6 flow

Manufactured by BD

Sourced in United States

The Accuri C6 flow cytometer is a compact and easy-to-use instrument designed for cell analysis and sorting. It provides high-performance flow cytometry capabilities in a benchtop format. The Accuri C6 is capable of detecting and analyzing a wide range of cell types, including mammalian cells, bacteria, and microparticles. It offers multiple fluorescence and light scatter detection channels to enable comprehensive cell characterization.

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Close spelling variants of this product

BD Accuri™ C6 Flow Cytometry system BD Accuri C6 flow cytometer Accuri C6 flow cytometry Accuri C6 Flow analyzer Accuri C6 FACScan flow cytometer BD Accuri C6 flow cytometer instrument Accuri C6 flow cytometer and software Accuri C6 Flow Cytometer System Accuri’s C6 Flow Cytometer FACS ACCURI C6 flow cytometer

15 protocols using accuri c6 flow

Cell Cycle Analysis by Flow Cytometry

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Cell cycle analyses of fixed cells were performed using either the Accuri C6 flow cytometer or the Chemometec NC-3000 image cytometer depending on experimental design.
For NC-3000 assays, cells were fixed with 70% ethanol for 2 to 24 h at −20°C prior to permeabilization with 0.1% Triton X-100 and incubated with 1 μg/ml DAPI DNA stain for 5 min at 37°C. Exported FCS3 files were processed using Flowing software to delineate cell cycle phases and determine proportions of cells in each fraction.
For Accuri C6 flow cytometry, cells were fixed with 1% formaldehyde for 10 min at 37°C before being permeabilized with methanol (90% final concentration) at −20°C for 2 to 24 h. Cells were pelleted and washed with PBS before being incubated with 50 μM DRAQ5™ or 125 ng/ml DAPI DNA staining for 30–60 min in the dark at room temperature. More than 5000 events were collected with the Accuri C6 flow cytometer and cell cycle distributions were determined from histograms generated using the Accuri C6 analysis software.

Liddiard K., Ruis B., Kan Y., Cleal K., Ashelford K.E., Hendrickson E.A, & Baird D.M. (2018). DNA Ligase 1 is an essential mediator of sister chromatid telomere fusions in G2 cell cycle phase. Nucleic Acids Research, 47(5), 2402-2424.

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Platelet Activation Assay

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Example 2

Platelets were isolated from citrated platelet-rich plasmas from 2-3 Group 0 blood donors (to minimize inter-donor platelet variability) in the presence of prostaglandin E1. Reaction mixtures consisted of 1×10⁶platelets in phosphate-buffered saline (PBS)-1% bovine serum albumin (BSA) to which was added (1) 50 μg/ml PF4, (2) 50 μg/ml PF4 and high dose (HD) UFH, or (3) buffer for 20 minutes at room temperature. The amount of PF4 added was optimized to maximize platelet activation in the assay. Ten microliters of serum was then added to produce a final reaction volume of 50 μl. Human PF4 was purified as previously described.

After incubation for 60 minutes at room temperature, phycoerythrin (PE)-labeled p-selectin antibody (BD Biosciences, San Jose, Calif.), and Alexa Fluor 647-labeled anti-GPIIb antibody 290.5 were added. After 15 minutes, the mixture was diluted in 200 μl of PBS-1% BSA and platelet events were acquired in an Accuri C6 flow cytometer. Events were gated by GPIIb positivity and p-selectin PE median fluorescence intensity (MFI) was recorded.

US10215767B2. Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis (2019-02-26). Blood Center Research Foundation [US]. Inventors: Richard H. Aster [US], Daniel W. Bougie [US], Curtis Gerald Jones [US], Anand Padmanabhan [US].

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Binding Kinetics of Recombinant Galectins

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Fixed hemocytes or fixed P. marinus trophozoites were incubated with 3% BSA in HBS 1 h, and subsequently incubated with 0–100 μg/ml of rCvGal1 or rCvGal2 for 1 h, both at room temperature. The cells were washed three times, and stained with 100 μl of rabbit anti-CvGal1 or anti-CvGal2 IgG respectively for 1 h, followed by incubation with Alexa 488-labeled anti-rabbit IgG for 1 h, both at room temperature. After three washes in HBS, the stained cells were analyzed with Accuri C6 flow cytometer. For competition experiments, cells were pre-incubated with of 0–100 μg/ml of rCvGal1 or rCvGal2, anti-A, or anti-B antibody for 30 min before adding 2 μg/ml of rCvGal2 or rCvGal1 respectively, followed by antibody detection.

Feng C., Ghosh A., Amin M.N., Bachvaroff T.R., Tasumi S., Pasek M., Banerjee A., Shridhar S., Wang L.X., Bianchet M.A, & Vasta G.R. (2015). The Galectin CvGal2 from the Eastern Oyster (Crassostrea virginica) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric Perkinsus Species. Biochemistry, 54(30), 4711-4730.

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Cell Cycle Analysis of MCF-7 Cells

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Following a 72 h/37°C incubation with each agent or the combinations, the MCF-7 cells were harvested by trypsinization, centrifuged at 300 x g, washed in PBS supplemented with 5% FBS (PBS+FBS), and suspended in a small amount of PBS+FBS. Ice-cold 70% ethanol was then added in a drop-wise fashion and the cells were fixed for 3 h/−20°C. After fixation, the ethanol was removed by centrifugation and the cells were washed in PBS+FBS and resuspended in sodium citrate (4 mM) in PBS+FBS. The cells were then incubated with 200 µg/mL of RNAse A (Sigma) and 30 µg/mL of propidium iodide (PI) for 20 min/37°C. The cell cycle distributions were measured using an Accuri C6 flow cytometer with PI being excited at 488 nm, and fluorescence detected at 585 nm (FL-2). A total of 10,000 events were collected per analysis.

Potuckova E., Jansova H., Machacek M., Vavrova A., Haskova P., Tichotova L., Richardson V., Kalinowski D.S., Richardson D.R, & Simunek T. (2014). Quantitative Analysis of the Anti-Proliferative Activity of Combinations of Selected Iron-Chelating Agents and Clinically Used Anti-Neoplastic Drugs. PLoS ONE, 9(2), e88754.

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Apoptotic Body Uptake by Macrophages

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We generated fluorescent apoptotic bodies by treating CellTracker Deep Red–labeled (Invitrogen) ES2 cells with 10 mM trametinib for 48 hours. Supernatants were harvested and apoptotic bodies were purified by sequential centrifugation for 10 min at 300g and then 10 min at 2000g (62 ). Apoptotic bodies were resuspended in fresh medium and incubated with pretreated M2-MΦ (100 nM trametinib or DMSO control for 24 hours) for 4 hours. MΦs were detached with 10 mM EDTA, stained with propidium iodide for viability, and then analyzed with an Accuri C6 flow cytometer to assess uptake of CellTracker-labeled apoptotic bodies.

Wang S.J., Li R., Ng T.S., Luthria G., Oudin M.J., Prytyskach M., Kohler R.H., Weissleder R., Lauffenburger D.A, & Miller M.A. (2020). Efficient blockade of locally reciprocated tumor-macrophage signaling using a TAM-avid nanotherapy. Science Advances, 6(21), eaaz8521.

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Apoptosis in Prostate Cancer Cells

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Apoptosis is a form of cell death that plays an important role during development, normal tissue homeostasis and is deregulated in many diseases, including cancer. The effect of apoptosis in LNCap prostate cancer cell lines was studied by flow cytometry. The early stages of apoptosis were detected following staining with a PE Annexin V Apoptosis Detection Kit I containing 7AAD as vital stain. The cells were seeded in 24 well flat-bottom plates at a cell density of 1 × 10 6 cells/well and incubated for 24 hr. SLN formulations, (empty, loaded) and pure RTA (25 ug/ml) were added to each well. Untreated cells were used as negative controls. After 48 hr, cells were stained according to the manufacturer"s protocol.
Samples were analysed on by using a Accuri C6 flow cytometer for PE and 7AAD expression using a solid state blue laser with a 488 nm excitation spectrum and a detector of FL1 path with a 530/30 nm filter. A minimum of 10000-gated events was acquired from the cell population and data analysed using the Accuri C6 software. Cells were considered early apoptotic when PE positive and 7AAD negative; late apoptotic/early necrotic when PE positive and 7AAD positive. All experiments were performed in triplicate and repeated three times.

Akanda M.H., Rai R., Slipper I.J., Chowdhry B.Z., Lamprou D., Getti G, & Douroumis D. (2015). Delivery of retinoic acid to LNCap human prostate cancer cells using solid lipid nanoparticles. International journal of pharmaceutics, 493(1-2).

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ALDH Activity Assay in MDA-MB-453 Cells

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The assay was performed as per manufacturer's protocol. 1 × 10 6 MDA-MB-453 cells were centrifuged and resuspended in 1 mL ALDH assay buffer. Five microliters of substrate were added into the cell suspension. DEAB was used as negative control. Cells were then incubated for 40 min at 37°C. Percentage of ALDH + cells were analyzed with Accuri C6 flow cytometer and Flowjo software.

López-Ozuna V.M., Hachim I.Y., Hachim M.Y., Lebrun J.J, & Ali S. (2019). Prolactin modulates TNBC aggressive phenotype limiting tumorigenesis. Endocrine-related cancer, 26(3).

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Protective Effects of THSG Against Gentamicin-Induced Apoptosis

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The UB/OC-2 cells in 6-well plates (4 × 10⁵ cells/well) were treated with various concentrations of THSG (5, 10, and 20 μM) for 6 h and subsequently with 750 μM gentamicin for 24 h. After collection, the cells were incubated with saturating concentrations of Annexin V-FITC and PI (2 μg/mL) in binding buffer for 15 min at room temperature. The results were analyzed by the BD Accuri™ C6 flow cytometry system.

Wen Y.H., Lin J.N., Wu R.S., Yu S.H., Hsu C.J., Tseng G.F, & Wu H.P. (2020). Otoprotective Effect of 2,3,4′,5-Tetrahydroxystilbene-2-O-β-d-Glucoside on Gentamicin-Induced Apoptosis in Mouse Cochlear UB/OC-2 Cells. Molecules, 25(13), 3070.

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Doxorubicin Uptake and Retention

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20,000 cells were seeded into 48-well plates and allowed to adhere for 24 h at 37 °C in a 5% CO2 and 95% air humidified incubator. The concentration of doxorubicin or pDox was kept at 5 µM for doxorubicin control, DNPs and VDNPs. DNPs were either incubated alone, or with VNPs dose-matched at the concentration of VES-H₈R₈ in VDNPs. To test Pgp inhibition, DNPs were also incubated with 20 µM of vitamin E succinate. Untreated cells were used as negative control to determine background. After 24 hours of treatment, cells were washed thrice with PBS, and harvested with trypsin. Cell fluorescence was analyzed using a BD Accuri C6 flow. Cell debris and doublets were gated out using FSH-A vs FSH-H, and at least 10,000 events were collected. The mean fluorescence intensity was measured on three biological repeats to evaluate the doxorubicin or pDox uptake and retention.

Czupiel P., Delplace V, & Shoichet M. (2020). Nanoparticle delivery of a pH-sensitive prodrug of doxorubicin and a mitochondrial targeting VES-H8R8 synergistically kill multi-drug resistant breast cancer cells. Scientific Reports, 10, 8726.

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CD44 Expression Analysis by Flow Cytometry

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The protein expression level of CD44 was analyzed using BD Accuri C6 flow cytometer (BD Biosciences). Trypsinized cells (10⁵–10⁸ cells/ml) were suspended in complete medium and stained with 31.3 ng/ml CD44 monoclonal antibody (IM7) PE-Cyanine5 (#15-0441-82, Gibco, Thermo Fisher Scientific) for 30 min at room temperature. Then, cells were washed twice and finally resuspended in Hanks’ balanced salt solution (Gibco, Thermo Fisher Scientific). Cell aggregates were removed by filtering using a 70-µm cell strainer (BD Biosciences) before flow cytometry analysis. Unstained control cells were used to discriminate positively stained cells.

Dolatabadi S., Jonasson E., Andersson L., Luna Santamaría M., Lindén M., Österlund T., Åman P, & Ståhlberg A. (2022). FUS-DDIT3 Fusion Oncoprotein Expression Affects JAK-STAT Signaling in Myxoid Liposarcoma. Frontiers in Oncology, 12, 816894.

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