Novocyte flow cytometer system

Manufactured by Agilent Technologies

Sourced in United States

The NovoCyte Flow Cytometer Systems by Agilent Technologies are analytical instruments designed to perform multiparameter analysis of cells and particles in a fluid sample. These systems utilize flow cytometry technology to rapidly measure and analyze the physical and fluorescent characteristics of individual cells or particles as they pass through a laser beam.

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13 protocols using novocyte flow cytometer system

Flow Cytometry Analysis of Cell Signaling

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Cells were trypsinized and resuspended to a final concentration of 2 million cells/mL. For each condition, 400′000 cells (200 µl) were distributed in flow cytometry tubes and OGG1i or pump inhibiting drugs were added at a 2X of final concentration. On top of that, 200 µl of warm medium containing the investigated dye at 2X of final concentration (MitoTracker Green 100nM, TMRE 100nM, Sir-DNA 333nM, Hoechst33342 0.1 μg/μl, and Mitoxantrone 1 µM) was added. Cells were incubated 30 min at 37°C and further 10 min at room temperature for equilibration before flow cytometry analysis with the NovoCyte Flow Cytometer System (Agilent).
The immunostaining for Phospho-H3 was performed in cells resuspended, fixed and permeabilized in ice-cold 75% ethanol. Upon washing, cells were blocked in PBS buffer containing 1% BSA, 0.05% Triton X100 and cells were incubated overnight at 4°C with an Pacific Blue Conjugate monoclonal antibody against Phospho-Histone H3 (Ser10) (8552, Cell Signaling) diluted at 1:500 in PBS buffer containing 1% BSA, 0.05% Triton X100. Washed cells were resuspended in PBS-1% BSA in the presence of 0.3 µM of Topro-3 for DNA staining 1 h at room temperature before FACS analysis with the NovoCyte Flow Cytometer System (Agilent).

Tanushi X., Pinna G., Vandamme M., Siberchicot C., D’Augustin O., Di Guilmi A.M., Radicella J.P., Castaing B., Smith R., Huet S., Leteurtre F, & Campalans A. (2023). OGG1 competitive inhibitors show important off-target effects by directly inhibiting efflux pumps and disturbing mitotic progression. Frontiers in Cell and Developmental Biology, 11, 1124960.

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Annexin V-FITC/PI Apoptosis Assay

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Apoptosis was detected using flow cytometry using Annexin V-FITC/PI Apoptosis kit. A total of 1-3x10⁶ cells were pelleted and washed twice with 1 ml PBS, resuspended in 300 µl pre-chilled binding buffer, and then 3 µl annexin V-FITC and 5 µl PI-PE were added to cells. After gentle mixing, the cells were incubated at room temperature in the dark for 10 min, then loaded with 200 µl precooled binding buffer into the cytometer for analysis according to the manufacturer's instructions (NovoCyte Flow Cytometer System; Agilent Technologies, Inc.) using NovoExpress (v.6.2; Agilent Technologies, Inc.).

Wang X., Zhang Y., Wuyun K, & Gong H. (2021). Therapeutic effect and mechanism of 4-phenyl butyric acid on renal ischemia-reperfusion injury in mice. Experimental and Therapeutic Medicine, 23(2), 144.

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Annexin V-FITC/PI Apoptosis Assay

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Cell apoptosis detection was performed using the Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN) according to the manufacturer’s manual. In brief, cells were resuspended in 500 μL Binding Buffer, stained with Annexin V-FITC and Propidium Iodide (PI) for 15 min in the dark, and analyzed using the NovoExpress software (version 1.4.1, Agilent, USA) with the NovoCyte Flow Cytometer System (Agilent). Gating strategy was shown in Supplementary Fig. 6.

Gong T.T., Liu F.H., Xiao Q., Li Y.Z., Wei Y.F., Xu H.L., Cao F., Sun M.L., Jiang F.L., Tao T., Ma Q.P., Qin X., Song Y., Gao S., Wu L., Zhao Y.H., Huang D.H, & Wu Q.J. (2024). SH3RF2 contributes to cisplatin resistance in ovarian cancer cells by promoting RBPMS degradation. Communications Biology, 7, 67.

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Multiparametric Profiling of Stem Cell Markers

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Trypsinized (Biological Industries, Cat: BI03-052-1B) cells were collected and washed with ice-cold PBS once. Next, cells were fixed with 4% Paraformaldehyde (Sigma, Cat:158127) in PBS for 20 minutes at room temperature and centrifuged at 1500 rpm for 5 minutes; following by resuspension in stain buffer (BD, Cat: 554656) in a concentration scale of 1 × 106 cells/ml. Following antibodies were used as described; EpCAM-APC (BD, 347200) (1:100 v/v), CD133-PE (BioLegend, 372804) (1:100 v/v), CD44-FITC (Miltenyi Biotec, Cat: 130-095-195) (1:10 v/v) and CD90-FITC (Miltenyi Biotec, Cat: 130-095-403) (1:10 v/v). For unstained controls, IgG1 Isotypes; IgG1-FITC (Immunostep, Cat: ICIGG1F-100), IgG1-PE (Immunostep, Cat: ICIGG1PE-50), and IgG1-APC (BD, Cat: 555751); were used as 1:20 (v/v). For staining, cells were incubated with antibodies for 30 minutes in room temperature at dark and washed once with staining buffer. Stained cells were analyzed on NovoCyte Flow Cytometer System (Acea) and analysis was performed via NovoExpress Software (Supplementary Fig. S1).

Sahin I.D., Christodoulou M.S., Guzelcan E.A., Koyas A., Karaca C., Passarella D, & Cetin-Atalay R. (2020). A small library of chalcones induce liver cancer cell death through Akt phosphorylation inhibition. Scientific Reports, 10, 11814.

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Flow Cytometry Analysis of Murine Immune Cells

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Animals were sacrificed and the spleens and kidneys were collected for flow cytometry analysis. The monoclonal antibodies used for splenic flow cytometry were CD4-PE, CD3-PE-cy7, Foxp3-FITC, CD69-percp-cy7, B220-PE-cy7, CD21-FITC, CD23-PE, CD11c-PE-cy7, CD11b-APC, F4/80-PE, CD86-FITC, and F4/80-PErCP. The monoclonal antibodies used for renal flow cytometry were CD4-PE, CD3-Percp, Foxp3-FITC, CD45-APC-cy7, CD11b-FITC, CD11c-PE-cy7, F4/80-PE, and Gr-1-Percp (eBioscience, Hanover Park, IL, USA). Cell counting was performed by using a Cellometer^® automated cell counting system (Sigma-Aldrich, St Louis, MO, USA) for absolute cell numbers. The Novocyte flow cytometer system (ACEA Bioscience Inc., San Diego, CA, USA) was used for flow cytometry, and analysis was performed as described [11 (link)]. Data were analyzed using Novocyte software (ACEA Bioscience Inc.). At least 200,000 events were acquired for each analysis.

Qin L., Du Y., Ding H., Haque A., Hicks J., Pedroza C, & Mohan C. (2019). Bradykinin 1 receptor blockade subdues systemic autoimmunity, renal inflammation, and blood pressure in murine lupus nephritis. Arthritis Research & Therapy, 21, 12.

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Flow Cytometric Analysis of Procyclin Expression

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To monitor the proportion of the population expressing EP and/or GPEET procyclins, flow cytometry was performed as described previously [19 (link),20 (link)]. The primary antibodies were anti-EP (mouse monoclonal TBRP1/247, Cedarlane Laboratories, Burlington, Canada) and anti-GPEET (rabbit polyclonal K1) [79 (link)] and the secondary antibodies were Alexa Fluor 488-conjugated goat anti-rabbit (Invitrogen, Eugene, Oregon, USA) and Cy3-conjugated goat anti-mouse (Jackson ImmunoResearch). Antibody dilutions are mentioned in S3 Table. Fluorescence of 20,000 cells was measured using a Novocyte flow cytometer system (ACEA Biosciences, Inc., San Diego, USA).

Bevkal S., Naguleswaran A., Rehmann R., Kaiser M., Heller M, & Roditi I. (2021). An Alba-domain protein required for proteome remodelling during trypanosome differentiation and host transition. PLoS Pathogens, 17(1), e1009239.

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Ascitic Fluid Analysis by Flow Cytometry

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Ascitic fluid from the NS, DOTAP, PST34A and PST34A+DOTAP treatment groups was collected. A total of 5 ml normal saline solution was injected into abdomen cavity and withdrawn from mice with ascitic fluid. All specimens were washed with PBS, resuspended in propidium iodide/RNase A solution (5 ml; Beyotime Institute of Biotechnology) and incubated in the dark at 4°C for 15 min. Samples were analyzed by flow cytometry using the NovoCyte^® Flow Cytometer System which included the analysis software (ACEA Biosciences, San Diego, CA, USA).

Qiu F, & Zhao X. (2018). In vivo antitumor activity of liposome-plasmid DNA encoding mutant survivin-T34A in cervical cancer. Molecular Medicine Reports, 18(1), 841-847.

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Apoptosis Evaluation of BV-2 Cells

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Cell apoptosis of BV-2 cells was measured by flow cytometry using the annexin V staining kit (BD Biosciences, San Jose, CA, United States). In brief, BV-2 cells were pretreated with 5 μM Aβ(1-42) for 12 h, then followed with an incubation of 0.12–0.48 mg⋅L^-1 LSF. After treatment, BV-2 cells were analyzed by the NovoCyte Flow Cytometer Systems (ACEA Biosciences) using FITC-annexin V and propidium iodide staining (BD Biosciences, San Jose, CA, United States) according to the manufacturer’s instructions. Data acquisition and analysis were performed with NovoExpress software (ACEA Biosciences).

Zhao Y., Zeng Y., Wu A., Yu C., Tang Y., Wang X., Xiong R., Chen H., Wu J, & Qin D. (2018). Lychee Seed Fraction Inhibits Aβ(1-42)-Induced Neuroinflammation in BV-2 Cells via NF-κB Signaling Pathway. Frontiers in Pharmacology, 9, 380.

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Annexin V-FITC Apoptosis Assay

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For the detection of cell apoptosis, cells were harvested by trypsin without ethylenediaminetetraacetic acid (EDTA) and washed with PBS. Then, 380 μL of 1 × binding buffer was added to each sample containing at least 0.5 × 10⁶ cells. Then, AV-FITC apoptosis detection assay reagent (lot#: 185174000, Invitrogen; 10 μL of FITC annexin V and 5 μL of the PI working solution) was added to each sample for 15–30 min at room temperature. Apoptotic cells were analyzed based on AV and PI signals via NovoCyte Flow Cytometer Systems (ACEA Biosciences).

Lei H., Wang Z., Jiang D., Liu F., Liu M., Lei X., Yang Y., He B., Yan M., Huang H., Liu Q, & Pang J. (2021). CRISPR screening identifies CDK12 as a conservative vulnerability of prostate cancer. Cell Death & Disease, 12(8), 740.

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Apoptosis and Cell Cycle Analysis Protocol

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For apoptosis analyses, the cells were treated with candidate drugs for 72 hours. The attached cells were trypsinized and collected together with the supernatant. All the cells were washed twice with PBS and stained using the Annexin V‐FITC Apoptosis Detection Kit (TransGen, Beijing, China). PI and FITC channels of CytoFLEX Platform (Beckman Coulter, Inc., Brea, CA, USA) were used to detect apoptosis in the samples. For cell cycle analysis, the cells were trypsinized and fixed with 70% alcohol overnight. A total of 1 × 10⁶ fixed cells per sample were washed with PBS and stained with 50 μg/mL propidium iodide, 100 μg/mL RNase A, 0.2% Triton X‐100 in PBS solution. The cells were subsequently subjected to NovoCyte Flow Cytometer Systems (ACEA Biosciences, Inc., San Diego, CA, USA).

Chen A., Wen S., Liu F., Zhang Z., Liu M., Wu Y., He B., Yan M., Kang T., Lam E.W., Wang Z, & Liu Q. (2021). CRISPR/Cas9 screening identifies a kinetochore‐microtubule dependent mechanism for Aurora‐A inhibitor resistance in breast cancer. Cancer Communications, 41(2), 121-139.

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