Attune flow cytometer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom, Switzerland

The Attune flow cytometer is a high-performance instrument designed for cell analysis and sorting. It utilizes flow cytometry technology to provide accurate and efficient measurements of various cellular properties, including size, granularity, and fluorescence. The Attune flow cytometer is capable of analyzing a wide range of sample types and is suitable for a variety of applications in research and clinical settings.

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Lab products found in correlation

Apc brdu flow kit, by BD (5 mentions) Annexin 5 binding buffer, by Thermo Fisher Scientific (4 mentions) Fcs express 7, by De Novo Software (4 mentions) Flojo, by Tree Star (3 mentions) Permeabilization buffer, by Thermo Fisher Scientific (3 mentions) Propidium iodide, by Merck Group (3 mentions) Mouse primary antibody, by Thermo Fisher Scientific (3 mentions) Attune cytometric software v 1, by Thermo Fisher Scientific (3 mentions) Attune software, by Thermo Fisher Scientific (3 mentions) Software, by FlowJo (3 mentions) Ack lysis buffer, by Thermo Fisher Scientific (3 mentions) Sybr green 1, by Thermo Fisher Scientific (3 mentions) C6 flow cytometer, by BD (3 mentions) Anti cd81, by Santa Cruz Biotechnology (2 mentions) Anti cd63, by Santa Cruz Biotechnology (2 mentions) Collagenase enzyme, by Thermo Fisher Scientific (2 mentions) Coulter act diff analyzer, by Beckman Coulter (2 mentions) Eosin 5 maleimide, by Thermo Fisher Scientific (2 mentions) Facscalibur, by BD (2 mentions) Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (2 mentions)

231 protocols using attune flow cytometer

Extracellular and Intracellular Staining for Flow Cytometry

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Extracellular staining was carried out to detect β2m and CXCR4. Briefly, 3 × 10⁵ to 1 × 10⁶ cells were washed with 2% BSA in PBS (FLUO-Buffer). To determine the living cell population, cells were incubated with Fixable Viability Dye eFluor 780 (eBioscience) at a dilution of 1:1,000. After washing with FLUO-Buffer, primary antibodies (concentration shown in Table 2) were applied for 20 min. Cells were washed in FLUO-Buffer to remove unbound antibodies and incubated with conjugated secondary antibodies for 20 min. Subsequently, cells were again washed and analyzed using an Attune flow cytometer (Thermo Fisher Scientific). To detect CD45+ cells, a direct fluorescein isothiocyanate (FITC)-labeled primary antibody was used, and the cells were washed with FLUO-Buffer before analysis. Antibodies and concentrations used are shown in Table 2.
For intracellular staining, cells were fixed and permeabilized with 0.1% Triton X-100 in PBS for 10 min. The subsequent staining process was carried out as described above. Detection of EGFP signal loss in different cells was examined by quantifying the loss of EGFP signal. For all flow cytometry analyses, an Attune flow cytometer (Thermo Fisher Scientific) was used. Data were analyzed with FlowJo 10.4.1 software (FlowJo, LLC 2006-2017).

Rieblinger B., Sid H., Duda D., Bozoglu T., Klinger R., Schlickenrieder A., Lengyel K., Flisikowski K., Flisikowska T., Simm N., Grodziecki A., Perleberg C., Bähr A., Carrier L., Kurome M., Zakhartchenko V., Kessler B., Wolf E., Kettler L., Luksch H., Hagag I.T., Wise D., Kaufman J., Kaufer B.B., Kupatt C., Schnieke A, & Schusser B. (2021). Cas9-expressing chickens and pigs as resources for genome editing in livestock. Proceedings of the National Academy of Sciences of the United States of America, 118(10), e2022562118.

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Apoptosis and Cell Cycle Analysis

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For apoptosis analysis, 1.5 × 10⁵ cells/well were seeded in 6-well plates. After overnight incubation, the cells were treated with or without quercetin (50, 100, 200 μM) and luteolin (10, 20, 40 μM) for 48 h. The cells were collected and suspended in a 100 μl binding buffer containing 5 μl FITC-conjugated Annexin V and 5 μl PI (Annexin V FITC Apop Dtec, BD). After dark incubation for 15 min at room temperature, a 400 μl binding buffer was added. The apoptosis condition of the cell samples was analyzed using the Attune Flow cytometer (ThermoFisher, USA) within 1 h.
For cell cycle analysis, the cells were collected and fixed with 75% ethanol at −20°C overnight. Then, the cells were incubated with 0.5 ml propidium iodide (PI, 0.5 ml FxCycleTM PI/RNAse Solution, BD) in the dark for 45 min. The cell samples were tested using the Attune Flow cytometer (ThermoFisher, USA) for cell cycle analysis.

Chen Y., Hao E., Zhang F., Du Z., Xie J., Chen F., Yu C., Hou X, & Deng J. (2021). Identifying Active Compounds and Mechanism of Camellia nitidissima Chi on Anti-Colon Cancer by Network Pharmacology and Experimental Validation. Evidence-based Complementary and Alternative Medicine : eCAM, 2021, 7169211.

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Competitive Growth Assay of APEX1/2 KO Cells

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WT or TDP1-KO cells were infected with virus particles expressing plenti-V2-RFP-sgAAVS1 (control) or a plenti-V2-GFP-sgRNA targeting APEX1 or APEX2. Seventy-two hours after infection, RFP- and GFP-expressing cells were mixed 1:3. A fraction of mixed cells were seeded into plates, and the remaining mixed cells were analyzed by Attune Flow cytometers (ThermoFisher). During the course of the experiments, cells were sub-cultured at the indicated time points and analyzed by Attune Flow cytometers (ThermoFisher). The day of the mixture was set as day 0. Collected data were analyzed with FlowJo software (FlowJo 10.6.1, Becton Dickinson), and the relative ratios of GFP- and RFP-positive cells were calculated.

Zhang H., Xiong Y., Su D., Wang C., Srivastava M., Tang M., Feng X., Huang M., Chen Z, & Chen J. (2022). TDP1-independent pathways in the process and repair of TOP1-induced DNA damage. Nature Communications, 13, 4240.

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Liver Tissue Extraction and Cell Cycle Analysis

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Liver tissues from mothers and fetuses of all groups were weighed, rinsed in phosphate-buffered saline, and minced using a pair of scissors into 1-mm fragments then digested using collagenase (1 ml/0.25 g tissue) (Meng et al., 2016; (link)Seglen, 1976) (link). Cell cycle analysis by flow cytometry through PI staining was performed according to the method previously described by Allen and Davies (2007) (link). Attune flow cytometer (Applied Biosystem, USA) was used to analyze the stained cells.

Basal W., Omar A.R., & Mahmoud A. (2021). Exposure to Lufenuron During the Third Gestational Period Induces Genotoxicity and Oxidative Stress Effects in Pregnant Albino Rats and Their Fetuses. Egyptian Academic Journal of Biological Sciences. B, Zoology, 13(2), 121-134.

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Cell Cycle Analysis of HL60/RS Cells

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The harvested HL60/RS cells were fixed in ice-cold 70% ethanol at -20 °C for 24 h after trypsinization. Following twice PBS washes, the cells were stained with 100 µl propidium iodide (PI) and incubated in darkness for 1 h. The stained cells were analyzed by Attune flow cytometer (Applied Bio-system, US).

Othman R.Q.A., Badawy A., Alruwaili M., & El‐Magd M.A. (2021). Camel Milk Exosomes Potentiate The Anticancer Effect of Doxorubicin on Multidrug-Resistant Human Leukemia Hl60 Cells in Vitro and in Vivo.

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Autophagosome Detection in EAC Cells

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A flow cytometer was used for the detection of EAC fluorescent autophagosomes. A proprietary fluorescent autophagosome marker (λex = 333/λem = 518 nm) was utilized within an autophagy assay kit (Sigma Aldrich, USA). One hundred microliters of each ascetic fluid of EAC-bearing control mice and treated groups was added to 1 mL of phosphate-buffered saline then centrifuged for 5 min at 21 °C. An autophagosome detection reagent (4,6-diamidino-2-phenylindole (DAPI stain)) was diluted with Dulbecco’s Modified Eagle Medium at 1/10 dilution, and then 4 µL of this reagent was added to cells in each well and the mixture was incubated at 37 °C with 5% CO2 for 30 min. The excess dye was removed by washing with a wash buffer and then centrifuged for 5 min at 21 °C for removal of the excess dye. The washing step was repeated twice.
One hundred microliters of phosphate-buffered saline was added to the last pellet and the fluorescence was determined (Ex. 360 nm, Em. 520 nm), where ex: fluorescent excitation (blue), em: fluorescent emission (green) in blue configuration, with an Attune Flow Cytometer (Applied Biosystem, USA).

Tayel F., Mahfouz M.E., Salama A.F, & Mansour M.A. (2021). Ethoxyquin Inhibits the Progression of Murine Ehrlich Ascites Carcinoma through the Inhibition of Autophagy and LDH. Biomedicines, 9(11), 1526.

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Quantifying Apoptosis in Cancer Cells

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Apoptotic cells were quantified using an Annexin V-FITC-propidium iodide (PI) double staining kit (Cat. No. 556547 BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s instructions. Each of the HCT-116 and Caco-2 cells was plated in a 24-well plate (5 ´ 10⁵ cells/well) and then incubated for 24 hours for attachment, and then the cells were treated with Q and/or 5-FU, as in the experimental design, for 48 hours. After cell collection by trypsinization, HCT-116 and Caco-2 cells were washed with PBS and stained by annexin V/propidium (BD Biosciences) based on the manufacturer’s instructions for 25 minutes at room temperature in a dark place. The stained cells were analysed using an Attune flow cytometer (Applied Bio-system, USA). Experiments were performed in triplicate.

Terana G.T., Abd-Alhaseeb M.M., Omran G.A, & Okda T.M. (2022). Quercetin potentiates 5-fluorouracil effects in human colon cancer cells through targeting the Wnt/β-catenin signalling pathway: the role of miR-27a. Contemporary Oncology, 26(3), 229-238.

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Ru(III) Complex Cytotoxicity Assay

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The Ru(III) complex (2) (117.6 μg/mL) was added to MCF7 and Caco2 cells at a confluence of 70–80% and incubated for 24 h (in a CO₂ incubator). The cells were then digested with warm trypsin-EDTA + warm PBS-EDTA (0.25%) (500 + 500 μL) with incubation for 10 min at 37 °C. The mixture was centrifuged (450 rpm) for 5 min, then the supernatant was removed. The mixture was washed twice in warm PBS and the cell pellet was re-suspended in 500 µl warm PBS, centrifuged and the supernatant decanted. A volume of 150 μL PBS + 350 μL ice-cold 70% ethanol was added and incubated at 4 °C for 1 h to fix the cells. To remove ethanol, the mixture was centrifuged at 350 rpm for 10 min and then the supernatant was removed. The mixture was washed twice in warm PBS and the cells were re-suspended in 500 μL of warm PBS, centrifuged, and the supernatant was removed. The cells were resuspended in 100 μL PBS and stored at 4 °C for up to four days. In darkness, the cells were stained with 100 µL of propidium iodide (PI) solution + 50 µL RNase A solution (100 µg/mL) and incubated in darkness for 30–60 min. The stained cells were read in an Attune flow cytometer (Applied Bio-system, Foster City, CA, USA).

Elsayed S.A., Harrypersad S., Sahyon H.A., El-Magd M.A, & Walsby C.J. (2020). Ruthenium(II)/(III) DMSO-Based Complexes of 2-Aminophenyl Benzimidazole with In Vitro and In Vivo Anticancer Activity. Molecules, 25(18), 4284.

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Isolation and Characterization of Milk Exosomes

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Differential ultracentrifugation was used to isolate the exosomes. Fat globules, casein aggregates, and other debris were removed from the milk sample by centrifugation at 5000 g for 15 min at 4 °C then at 13,000 g/30 min/4 °C. Exosomes were extracted from supernatants by twice ultracentrifugation at 100,000 g (Optima L-90okay; Beckman Coulter) for 90 min each at 4 °C, followed by a brief wash with phosphate-buffered saline (PBS) to remove large debris and microvesicles. To produce a homogeneous suspension, the exosome pellets were collected and resuspended in PBS at a concentration of 6 mg/mL, centrifuged at 5000 rpm for 30 min twice with the 100 kDa filters, and stored at 4 °C until the next use. Transmission electron microscopy (TEM) at 80 kV was used to identify the size of exosomes as previously described [27 (link)]. Confirmation of exosomal isolation was carried out by the detection of specific exosomal proteins CD63 and CD81 using anti-CD63 (1:200, Santa Cruz, Germany) and anti-CD81 (1:200, Santa Cruz, Germany) by Attune flow cytometer (Applied Biosystem, Foster City, CA, USA) and a standard Nanobead calibration kit containing beads (50 and 100 nm, Technologies Drive Fisher, Ann Arbor, MI, USA).

Shaban A.M., Raslan M., Sharawi Z.W., Abdelhameed M.S., Hammouda O., El-Masry H.M., Elsayed K.N, & El-Magd M.A. (2023). Antibacterial, Antifungal, and Anticancer Effects of Camel Milk Exosomes: An In Vitro Study. Veterinary Sciences, 10(2), 124.

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Cell Cycle Analysis of Liver Tissue

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Flow cytometry was used to determine the influence of various treatments on the number of cells in each phase of the cell cycle, and it was carried out as previously described by Abdelwahab et al. (2019) (link) with minor changes. Collagenase enzyme (0.1 percent, Invitrogen, United States) was used to lyse freshly dissected liver tissues, which were subsequently mesh filtered (0.7 mm nylon) and centrifuged (4,000 rpm/5 min). The cells were fixed and stained with propidium iodide before being examined with an Attune flow cytometer (Applied Bio-system, United States). Each cell cycle phase’s number of cells was counted and expressed as a percentage of the total number of cells.

Jebur A.B., El-Sayed R.A, & El-Demerdash F.M. (2022). Ocimum basilicum Essential Oil Modulates Hematotoxicity, Oxidative Stress, DNA Damage, and Cell Cycle Arrest Induced by β-cyfluthrin in Rat Liver. Frontiers in Pharmacology, 12, 784281.

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