Novocyte cytometer

Manufactured by Agilent Technologies

Sourced in United States

The Novocyte cytometer is a flow cytometry instrument designed for cell analysis. It provides the core function of detecting and analyzing cells in a sample by measuring their physical and fluorescent properties as they pass through a laser beam.

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Agilent NovoCyte flow cytometer NovoCyte Quanteon Flow Cytometer NovoCyte Advanteon B4 Flow Cytometer 3-Laser Novocyte Flow Cytometer 6-color Novocyte Cytometer Novocyte 3000 flow cytometer NovoCyte 2000 R Flow Cytometer NovoCyte 2040R flow cytometer ACEA Novocyte™ flow cytometer Agilent NovoCyte Quanteon flow cytometer

13 protocols using novocyte cytometer

Identification of DC Subsets and Monocytes

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Human peripheral blood mononuclear cells were isolated via Lymphoprep density gradient centrifugation from whole blood (StemCell; Cambridge, MA) (Cat# 07801). For identification of peripheral CD123⁺DCs and basophils, the following staining panel was used: FITC anti-human Lin [CD3(UCHT1), CD14(HCD14), CD16(3G8), CD19(HIB19), CD20(2H7), CD56(HCD56)], PE anti-human LMAN1 (OTI1A8), PerCP anti-human HLA-DR (L243), PE/Cy7 anti-mouse CD11c (S-HCL-3), APC anti-human CD123 (6H6). For identification of monocyte populations, the following staining panel was used: FITC anti-human Lin [CD3(SK7), CD19(4G7), CD20(2H7), CD56(HCD56)], PE anti-human LMAN1 (OTI1A8), PerCP anti-human HLA-DR (L243), PE/Cy7 anti-mouse CD11c (S-HCL-3), APC anti-human CD14 (M5E2), APC/Cy7 anti-human CD16(3G8). FMO controls excluded PE anti-human LMAN1 antibody. All cells were blocked prior to staining using Human TruStain FcX. All samples were acquired using a 6-color Novocyte cytometer (Agilent; Santa Clara, CA) (Cat# 2010049) and analyzed using NovoExpress software.

Miller M.H., Swaby L.G., Vailoces V.S., LaFratta M., Zhang Y., Zhu X., Hitchcock D.J., Jewett T.J., Zhang B, & Tigno-Aranjuez J.T. (2023). LMAN1 is a receptor for house dust mite allergens. Cell reports, 42(3), 112208.

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Annexin V-FITC Apoptosis Assay

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After digestion, the cells were collected. Cold PBS was used to wash cells for 3 times. The cells were centrifuged at 1500 g/min for 20 min. Cells were incubated with Propidium iodide (Beyotime, Shanghai, China) and Annexin V-FITC (Beyotime, Shanghai, China) in the dark for half hour. Cell cytometry was used to analyze cell apoptosis. Flow cytometry analysis (Agilent, Novocyte cytometer, USA) was used to analyze cell apoptosis. Flowjo™ software v11 was used for flow cytometry data analysis.

Lei Q., Wang Y., Li J., Wang S., Hu Y., Duan L., Huo Y., Wu Y, & Liu H. (2023). Increased SEMA6B expression as a potential prognostic and immune cell infiltration biomarker in thyroid cancer patients. Aging (Albany NY), 15(9), 3572-3585.

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Flow Cytometry Quantification of Claudin-1

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Cells detached with Trypsin-EDTA (Gibco) or with 10 mM EDTA (Invitrogen) for plasma membrane protein detection, were centrifuged at 450 g for 5 min at RT, washed once with PBS (Gibco) and fixed for 20 min at RT with 4% paraformaldehyde (PFA). Cells in suspension were incubated in blocking/permeabilization buffer (0.1% Triton X-100 and 0.5% BSA in PBS) for 30 min at RT. Cells were incubated with primary antibodies 2 h at RT, followed by washes and incubation with fluorophore-conjugated secondary antibodies diluted in blocking buffer for 45 min at RT.
For detection of the amount of total TagRFP-CLDN1 following drug treatment, cells were detached with 5 mM EDTA (Invitrogen) for 3 min. Cells were centrifuged at 800 g for 5 min and fixed with 4% PFA for 20 min at room temperature. Cells were washed three times with PBS and they were resuspended in PBS containing 0.5% BSA and 0.5 mM EDTA.
The percentage of positive cells and the mean fluorescence intensity (MFI) were determined by flow cytometry using a Novocyte cytometer (ACEA Biosciences) and results were analyzed using FlowJo (LLC) v10.

Clément C.M., Deffieu M.S., Dorobantu C.M., Baumert T.F., Ayala-Nunez N.V., Mély Y., Ronde P, & Gaudin R. (2020). Characterization of endogenous Claudin1 expression, motility and susceptibility to Hepatitis C Virus in CRISPR knock-in cells. Biology of the cell, 112(5), 140-151.

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Quantifying CD95 Cell Surface Expression

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To evaluate CD95 expression at the cell surface, cells (U87, RADH85 or RADH87) were seeded in 6 well plates (250000 cells/well). Twenty-four hours later, cells were harvested and stained using Zombie-violet^TM (Biolegend, 423113) diluted in PBS according to the manufacturer protocol. Then, samples were saturated with PBS containing 1% BSA and 1% FCS and human FcR-blocking reagent (Miltenyi Biotec, 130-059-901) for 15 min at 4 °C prior to labelling using an anti-human CD95-APC antibody (clone DX2, Miltenyi Biotec, 130-117-701, AB_2751411) or corresponding IgG1-APC isotype (clone IS5-21F5, Miltenyi Biotec, 130-113-196, AB_2733440) for 30 min at 4 °C. Following two washes with the saturation buffer, cells were resuspended in PBS and analysed using a Novocyte cytometer (Acea Biosciences). CD95 cell surface expression was analysed after gating on viable singlets. Results are represented as a ratio of Median Intensity Fluorescence values for anti-CD95-stained over isotype-stained samples.

Pelizzari-Raymundo D., Maltret V., Nivet M., Pineau R., Papaioannou A., Zhou X., Caradec F., Martin S., Le Gallo M., Avril T., Chevet E, & Lafont E. (2024). IRE1 RNase controls CD95-mediated cell death. EMBO Reports, 25(4), 1792-1813.

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Multiparameter Analysis of CAR T Cells

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CAR T cells were stained with a Live/Dead Fixable Near-IR stain (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min, followed by a phosphate-buffered saline (PBS) wash and incubation with a ChromPure Human IgG blocking solution (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 5 min. An antibody cocktail containing anti-PSMA antibody (clone LNI-17; BioLegend, San Diego, CA, USA), anti-EGFRt (Erbitux; Lilly, Indianapolis, IN, USA), anti-CD4 (clone OKT4; BioLegend), anti-CD8 (clone RPA-T8; BioLegend), and an in-house–generated anti-idiotypic antibody to detect the CAR was added for 20 min. Cells were then washed twice with staining buffer (BioLegend) and analyzed on a NovoCyte cytometer (ACEA Biosciences, San Diego, CA, USA). Data analysis was performed using FlowJo v10.1 software (Tree Star, Ashland, OR, USA).

Minn I., Huss D.J., Ahn H.H., Chinn T.M., Park A., Jones J., Brummet M., Rowe S.P., Sysa-Shah P., Du Y., Levitsky H.I, & Pomper M.G. (2019). Imaging CAR T cell therapy with PSMA-targeted positron emission tomography. Science Advances, 5(7), eaaw5096.

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Immunophenotyping Murine Immune Cells

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Mice were euthanised with CO₂, and blood was collected by cardiac puncture into EDTA-containing tubes (100 mmol/l EDTA). After lysing erythrocytes with RBC lysis buffer (eBiosciences, San Diego, USA) and washing with PBS, antibodies and viability dye were incubated with white blood cells in FACS buffer (PBS with 0.5% BSA and 2 mmol/l EDTA) for 20 min at 4°C. The following antibodies were used (Biolegend, San Diego, USA): CD45-FITC (30-F11 clone), Ly6G-Brilliant Violet 510 (1A8 clone), Ly6C-PE-Cy7 (HK1.4 clone), CD11b-APC (M1/70 clone), F4/80-PE (BM8 clone), CCR2-Brilliant Violet 421 (SA203G11 clone) and CD14-PE-Dazzle 594 (Sa14-2 clone). Viability dye (Fixable Viability Dye eFluor 780, eBiosciences) was added to distinguish live cells. Cells were washed in FACS buffer and fixed for 20 min at 4°C (Perm/Fix buffer, BD Biosciences, USA) before analysis on a Novocyte cytometer (Acea, USA).

Saadane A., Veenstra A.A., Minns M.S., Tang J., Du Y., Abubakr Elghazali F., Lessieur E.M., Pearlman E, & Kern T.S. (2023). CCR2-positive monocytes contribute to the pathogenesis of early diabetic retinopathy in mice. Diabetologia, 66(3), 590-602.

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Cellular Uptake of DNA/RNA Nanocubes

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PBMCs were either left untreated or incubated in the presence of DNA and RNA cubes alone, complexed with Lipofectamine, or complexed with G5 amine-terminated dendrimers. After 24 h of incubation, the cells were washed to remove the excess particles, reconstituted in the flow cytometry buffer, and analyzed using a Novocyte cytometer (ACEA Biosciences, San Diego, CA, USA). All cubes used for experiments were labeled with Alexa 488 (Integrated DNA technologies, Coralville, IA, USA ). The final particle concentration was 10 nM, the same as was used in the cytokine experiments. The cells were separated according to their forward and side scatter, and the live populations of lymphocytes and monocytes were gated into the green fluorescent channel for the detection of particle uptake. The data analysis was performed using the FCS Express software (DeNovo Software Inc., Pasadena, CA, USA).

Avila Y.I., Chandler M., Cedrone E., Newton H.S., Richardson M., Xu J., Clogston J.D., Liptrott N.J., Afonin K.A, & Dobrovolskaia M.A. (2021). Induction of Cytokines by Nucleic Acid Nanoparticles (NANPs) Depends on the Type of Delivery Carrier. Molecules, 26(3), 652.

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Murine Myeloid Cell Profiling

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Red blood cells were lysed using Lysing Buffer (Becton Dickinson, Italy) [54 (link)]. Monocytes and neutrophils counts were assessed by flow cytometry with a NovoCyte cytometer (ACEA) using the combination of the following antibodies: antiCD11b-PE, antiLy6C-FITC, and antiLy6G-PerCP antibodies from eBioscience [56 ]. A representative panel of the gating strategy is presented in the supplemental section.

Venu V.K., Moregola A., Da Dalt L., Uboldi P., Bonacina F., Muro A.F, & Norata G.D. (2023). Fibronectin extra domain a limits liver dysfunction and protects mice during acute inflammation. Atherosclerosis Plus, 52, 23-31.

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Annexin V-Light 650 and PI Assay

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Cells were extracted and washed twice with phosphate-buffered saline (PBS). Then those cells were coated in 500 µl of binding buffer containing 5 µl of Annexin V-Light 650 and 5 µl of propidium iodide (PI; Wanleibio) at room temperature in the dark for 15 min. Flow cytometry with a NovoCyte cytometer (ACEA Biosciences, San Diego, CA, USA) was performed to detect the stained cells.

Wei L., Wu Y., Cai S., Qin Y., Xing S, & Wang Z. (2023). Long Non-coding RNA Linc01224 Regulates Hypopharyngeal Squamous Cell Carcinoma Growth Through Interactions with miR-485-5p and IGF2BP3. Journal of Cancer, 14(16), 3009-3022.

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Isolation and Characterization of ILC2 cells

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Peripheral blood mononuclear cells (PMBC) and granulocytes were separated with Histopaque 1119 and Histopaque 1077 (Sigma Aldrich, St. Louis, MO) per manufacturers protocol. Nasal scrapings were processed through a strainer and washed prior to staining. Cells were first incubated with Fc Receptor Blocking Reagent (Miltenyi Biotec, San Diego, CA). Granulocytes were then stained with PerCP-conjugated CD45, FITC-conjugated FcεR1α, and APC-conjugated CCR3 (Biolegend, San Diego, CA). Eosinophils were defined as the percentage of CD45+ granulocytes that were CCR3+ FcεR1α−. PBMCs and nasal scrapings were stained with PerCP-conjugated CD45 (Biolegend), FITC-conjugated lineage markers (CD235a, FcεR1α, TCR γ/δ, CD4 (Biolegend), and BD Lineage Cocktail 1 [CD3, CD14, CD16, CD19, CD20, CD56]) and PE-conjugated CRTH2 (Miltenyi Biotec). PBMCs were also stained for APC-conjugated CD161 (Biolegend). Blood ILC2s were defined as CD45+ lineage negative CD161+ CRTH2+ lymphocytes as percentage of all cells. Percent of ILC2s are reported as percent of enriched PBMCs. Nasal scraping ILC2s were defined as CD45+ lineage-negative CRTH2+ lymphocytes. Percent of ILC2s are reported as percent of total cells in the nasal scraping sample. Flow cytometry was performed using a Novocyte cytometer (Acea Biosciences, Inc, San Diego, CA). Data were further analyzed with FlowJo software (FlowJo LLC, Ashland, OR).

Eastman J.J., Cavagnero K.J., Deconde A.S., Kim A.S., Karta M.R., Broide D.H., Zuraw B.L., White A.A., Christiansen S.C, & Doherty T.A. (2017). ILC2s are recruited to nasal mucosa in aspirin-exacerbated respiratory disease. The Journal of allergy and clinical immunology, 140(1), 101-108.e3.

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