Cytoflex lx flow cytometer

Manufactured by Beckman Coulter

Sourced in United States, Germany, China

The CytoFLEX LX is a flow cytometer designed for high-performance multiparameter analysis. It features a compact design and is capable of detecting up to 21 parameters simultaneously. The CytoFLEX LX utilizes a series of lasers and detectors to analyze the physical and fluorescent characteristics of cells or particles passing through the instrument.

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Lab products found in correlation

Cytexpert software, by Beckman Coulter (9 mentions) Kaluza analysis 2, by Beckman Coulter (5 mentions) Cytexpert, by Beckman Coulter (4 mentions) Alexa fluor 488 annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (3 mentions) Bovine serum albumin (bsa), by Merck Group (3 mentions) Cytexpert v2, by Beckman Coulter (3 mentions) Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (3 mentions) Cell staining buffer, by BioLegend (3 mentions) Cytofix cytoperm, by BD (3 mentions) Click s ehaa media, by Fujifilm (3 mentions) Dnase, by Worthington (3 mentions) Fitc annexin 5 apoptosis detection kit, by BD (3 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions) Aria 3 flow cytometer, by BD (2 mentions) Zombie aqua fixable viability dye, by BioLegend (2 mentions) Tumor dissociation kit, by Miltenyi Biotec (2 mentions) 7 aad, by BioLegend (2 mentions) Live dead fixable blue dead cell stain kit, by Thermo Fisher Scientific (2 mentions) Fcr blocking reagent, by Miltenyi Biotec (2 mentions) Fluorescein isothiocyanate (fitc), by BioLegend (2 mentions)

Spelling variants of this product

CytoFLEX (3035 citations) CytoFLEX flow cytometer (1941 citations) CytoFLEX LX (476 citations) CytoFLEX cytometer (182 citations) CytoFLEX LX cytometer (18 citations) LX flow cytometer (2 citations) Flow CytoFlex (2 citations)

201 protocols using cytoflex lx flow cytometer

Phenotypic analysis of thawed PBMC

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Thawed PBMC were washed twice with RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, 55 μM β-mercaptoethanol and 0.02 mg/ml DNAse. Thawed PBMC were stained with viability marker Promokine IR-840 (PromoCell GmbH, Heidelberg, Germany) for 20 min at room temperature in PBS. One million PBMC were washed with FACS buffer and stained with DuraClone IM B cells containing the following lyophilized directly conjugated mAbs: IgD-FITC, CD21-PE, CD19-ECD, CD27-PC7, CD24-APC, CD38-AF750, IgM-PB, CD45-KrO. Cells were washed with FACS buffer and acquired at Cytoflex LX flow cytometer (Beckman Coulter, Hialeah, FL). A minimum of 500,000 cells was acquired on a Cytoflex LX flow cytometer (Beckman Coulter).

De Biasi S., Mattioli M., Meschiari M., Lo Tartaro D., Paolini A., Borella R., Neroni A., Fidanza L., Busani S., Girardis M., Coppi F., Mattioli A.V., Guaraldi G., Mussini C., Cossarizza A, & Gibellini L. (2023). Prognostic immune markers identifying patients with severe COVID-19 who respond to tocilizumab. Frontiers in Immunology, 14, 1123807.

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Flow Cytometric Analysis of T Cell Subsets

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Timing: 1–3 h

These steps use the CytoFLEX LX flow cytometer (Beckman Coulter) to acquire the data for further analysis by FlowJo software v10 (TreeStar).

Alternatives: Depending on the accessibility, these steps can use other flow cytometer machines to acquire data according to manufactory protocols.

Before acquiring data, use the CytoFLEX daily QC fluorosphere beads to run a QC/standardization program to ensure the flow cytometer works properly.
Use single-color staining samples to set up an appropriate level of compensation and unstained samples to define the baseline level of auto-fluorescence for different fluorochromes on the flow cytometry machine used.

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Collect the data on the CytoFLEX LX flow cytometer (Beckman Coulter).

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When analyzing the data using FlowJo, gate the samples according to the gating strategy shown in Figure 4.Figure 4

Gating strategy for Th17, Treg, Th1, and Th2 cells

Representative plots depicting gating strategy of CD45+TCRβ+CD4+IL17A + Th17 cells, CD45+TCRβ+CD4+ GATA3+ Th2 cells, CD45+TCRβ+CD4+IFN-γ+ Th1 cells , and CD45+TCRβ+CD4+ FOXP3+ Treg cells. Adapted from Duan et al.¹ (link)

Duan J., Sun Y., Matute J.D, & Blumberg R.S. (2023). Characterizing CD4 T cell differentiation in mouse small intestine using T cell transfer, lamina propria preparation, and flow cytometry. STAR Protocols, 4(3), 102485.

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Quantification of HbF+ and Erythroid Differentiation

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For HbF^+ve cell analysis, 1 × 10⁵ erythroid differentiated cells were briefly washed with PBS and fixed with 0.05% glutaraldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 5 min. The cells were stained with anti-HbF APC antibody (dilution 1:50) and were acquired and analyzed using Cytoflex LX Flow Cytometer (Beckmann Coulter) or AriaIII flow cytometer (BD Biosciences) and analyzed using FlowJo (BD Biosciences). For erythroid differentiation analysis, 1 × 10⁵ cells from the terminal day of erythroid differentiation were stained for erythroid differentiation markers anti-CD71-FITC (dilution 1:33), anti-CD235a PE-Cy7 (dilution 1:50) and Hoechst 33342 (dilution 1:1,000). After 20 min of incubation in the dark, the cells were washed with PBS followed by analysis using Cytoflex LX Flow Cytometer (Beckmann Coulter) or AriaIII flow cytometer (BD Biosciences).

Venkatesan V., Christopher A.C., Rhiel M., Azhagiri M.K., Babu P., Walavalkar K., Saravanan B., Andrieux G., Rangaraj S., Srinivasan S., Karuppusamy K.V., Jacob A., Bagchi A., Pai A.A., Nakamura Y., Kurita R., Balasubramanian P., Pai R., Marepally S.K., Mohankumar K.M., Velayudhan S.R., Boerries M., Notani D., Cathomen T., Srivastava A, & Thangavel S. (2023). Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies. Molecular Therapy. Nucleic Acids, 32, 671-688.

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Quantification of NF-κB p52 Expression in CLL

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NF-κB p52 expression levels were quantified using a phycoerythrin-labeled anti-NF-κB p52 (Santa Cruz, Heidelberg, Germany). Briefly, aliquots of 1 × 10⁶ primary CLL cells were treated with or without CW15337 (0–5 μM) for 24 h. Subsequently, cells were labeled with CD5-FITC and CD19-APC prior to being fixed and permeabilized using fix/perm reagent (Biolegend). Then, 5 μL of anti-NF-κB p52 antibody was added to each aliquot of cells. After incubation for 10 min, cells were washed and then resuspended in 1% paraformaldehyde solution prior to being analyzed by flow cytometry using a CytoFLEX LX flow cytometer.

Burley T.A., Kennedy E., Broad G., Boyd M., Li D., Woo T., West C., Ladikou E.E., Ashworth I., Fegan C., Johnston R., Mitchell S., Mackay S.P., Pepper A.G, & Pepper C. (2022). Targeting the Non-Canonical NF-κB Pathway in Chronic Lymphocytic Leukemia and Multiple Myeloma. Cancers, 14(6), 1489.

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CHIR-AB1 Expression and IgY Phagocytosis

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HD11 cells stimulated for 24 h with different concentrations of LPS or inactivated IBV antigen (company B) were assessed for expression of CHIR-AB1 by subsequently staining the cells in 50 μL of FACS buffer with 1:20-diluted hybridoma supernatant containing mouse-anti-chicken CHIR-AB1 (clone 8D12, mouse IgG2b, gift from Thomas W. Göbel, LMU Munich, Munich, Germany) and 0.1 μg/mL allophycocyanin (APC)-labeled goat-anti-mouse IgG2a (SouthernBiotech, Birmingham, AL, USA) for 20 min at 4 °C, with two washing steps in FACS buffer in between. Next, the cells were washed once in FACS buffer and once in DPBS^−/− followed by staining in 50 μL of DPBS^−/− with 1:400 Zombie Aqua Fixable Viability Dye (BioLegend Inc., San Diego, CA, USA) for 20 min at 4 °C. Finally, the cells were washed once more in FACS buffer and resuspended in 200 μL of FACS buffer for analysis using the CytoFLEX LX flow cytometer.
To determine the involvement of CHIR-AB1 in the uptake of IgY-opsonized beads, different concentrations of mouse-anti-chicken CHIR-AB1 were administered to HD11 cells 10 min before addition of the beads to block interactions between CHIR-AB1 and IgY-opsonized beads. Subsequent steps were according to the phagocytosis assay as described.

van den Biggelaar R.H., van Eden W., Rutten V.P, & Jansen C.A. (2020). Nitric Oxide Production and Fc Receptor-Mediated Phagocytosis as Functional Readouts of Macrophage Activity upon Stimulation with Inactivated Poultry Vaccines in Vitro. Vaccines, 8(2), 332.

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Evaluating MEC-1 Cell Migration and Apoptosis

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To evaluate MEC-1 cell migration, transwell plates with a filter diameter of 6.5 mm and pore size of 5.0 μm were used. Cells were diluted to 1 × 10⁶ cells/mL in RPMI complete medium, with or without the addition of CW15337 (0, 2.5, 5, 10 μM). Subsequently, 5 × 10⁵ cells/well were seeded into the upper transwell insert. The lower compartment of the chamber was filled with 500 μL of complete medium supplemented with 100 ng/mL CXCL12. The chambers were incubated at 37 °C for 24 h and then removed from the plate. Cells that had migrated to the lower chamber were harvested and counted using a CytoFLEX LX flow cytometer. The level of apoptosis induced by CW15337 during the assay was quantified by harvesting cells from the upper chamber of the transwell (unmigrated cells), followed by labeling with Annexin V and 7-AAD (as above).

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Cytokine and Chemokine Profiling in Serum

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Serum was isolated by centrifugation, stored at −80°C, and thawed for cytokine assessment using BD Cytometric bead array human T_H1/T_H2 cytokine kit, human inflammatory cytokine kit, and human chemokine kit according to the manufacturer’s instructions. Samples were acquired on Cytoflex LX flow cytometer and analyzed by FlowJo 9.1 software (Treestar, Ashland, Ore). To evaluate CXCL9, IL-18, and IL-18 binding protein, commercially available enzyme-linked immunosorbent assays (ELISAs) were used according to the manufacturer’s instructions (catalog DY392, DY318-05, and DY119; R&D Systems, Minneapolis, Minn). Free IL-18 levels were calculated considering the law of mass action, as described elsewhere.²⁹
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Rey-Jurado E., Espinosa Y., Astudillo C., Jimena Cortés L., Hormazabal J., Noguera L.P., Cofré F., Piñera C., González R., Bataszew A., Muñoz Venturelli P., Benadof D., Álvarez P., Acevedo V., Vial P., Vial C, & Poli M.C. (2022). Deep immunophenotyping reveals biomarkers of multisystemic inflammatory syndrome in children in a Latin American cohort. The Journal of Allergy and Clinical Immunology, 150(5), 1074-1085.e11.

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Optimized Flow Cytometry for miRNA-MB Detection

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As the size of the streptavidin beads was 500 nm, the CytoFLEX LX flow cytometer was set up in the “nanoparticle detection mode” as previously reported(Camacho et al. 2017 ). Briefly, within the violet pod, the 450/45 bandpass was placed in position one and the 405/10 bandpass was placed in position two (Detector One). VSSC was used as the trigger parameter, and VSSCA linear versus SSCA log was plotted for bead population determination. The settings were optimized using Polysciences NIST Nanoparticle bead mix with sizes ranging from 80–500 nm, and set as follows: SSC: 58 V, VSSC: 50 V, FITC: 95 V, the FITC channel was used to measure the fluorescein fluorescence of the bead-attached MBs. For consistency, 50,000 events in the 500nm gate population were recorded for each specimen. Each sample was acquired at a rate of approximately 5,000 events per second. Quantification of miRNA-MB hybridization was measured by incubating at 37°C the MB-beads with 50 nM of synthetic miRNA oligonucleotide target analog and reading the samples by flow cytometry at the indicated times (0, 1, 5, 10, 20, and 30 min of incubation. The specificity of the interaction was determined for different hsa-miR-451a mismatch analog sequences (WT, and mutations 1, 2, 3, and 4, see Table 1). Target and mutation analog sequences were incubated at 55°C for 30 min before analysis.

Oliveira GP J.r., Barbosa R.H., Thompson L., Pinckney B., Murphy-Thornley M., Lu S., Jones J., Hansen C.H., Tigges J., Wong W.P, & Ghiran I.C. (2021). Electrophoretic mobility shift as a molecular beacon-based readout for miRNA detection. Biosensors & bioelectronics, 189, 113307.

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Live-cell Imaging and Flow Cytometry of Transgenic Parasites

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For live-cell imaging, parasite-infected blood was washed twice with PBS, followed by staining the cells with 1 μg/ml Hoechst 33342 (Sigma, Shanghai, China) in PBS. All images were captured and processed using identical settings in the OLYMPUS FRAME_BX63 scanning confocal microscope with a ×100 numerical-aperture (NA) oil objective.
To quantify the proportion of GFP-positive parasites, TPX-1 KO parasites were washed with PBS and analyzed with a flow cytometer. The cell nuclei were first stained with 2 μg/ml Hoechst 33342 (Sigma, Shanghai, China) and 2 μg/ml PI (propidium iodide) in PBS, with PI being used to count the dead cells. After a single wash, 100,000 cells were counted on the CytoFLEX LX flow cytometer, followed by selecting the cell populations without red fluorescent signal for data analysis using CytExpert 2.4, with gating for nuclear stain Hoechst 33342 and green fluorescence.

Wang S., Li D., Chen F., Jiang W., Luo W., Zhu G., Zhao J, & He L. (2022). Establishment of a Transient and Stable Transfection System for Babesia duncani Using a Homologous Recombination Strategy. Frontiers in Cellular and Infection Microbiology, 12, 844498.

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Flow Cytometric Analysis of Tumor-Infiltrating Cells

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Tumors were extracted and processed as described above before re-suspension in PBS buffer containing 2% FBS and PBS for flow cytometric analysis. Zombie NIR™ Fixable Viability Kit (Biolegend, USA) was applied to cells for 30 min on ice in the dark. Cells were washed and incubated with fluorochrome-conjugated antibody (anti-mouse CD45 Birliant violet 605, BioLegend; anti-mouse CD3 APC, BioLegend; anti-mouse CD8 FITC, BioLegend) at the manufacturer’s recommended dilution for 30 min on ice in the dark. For samples requiring intracellular staining, cells were fixed with Fixation/Permeablization Diluent (eBioscience cat. 00–5223-56) for 30 min at room temperature, washed twice with Permeablization Buffer (eBioscience cat. 00–8333-56), and incubated with antibody (anti-mouse Granzyme B PE, BioLegend; anti-mouse Ki-67 Alexa Fluor 700, BioLegend) in permeabilization buffer for 30 min at room temperature in the dark. Following staining, cells were washed again with permeabilizaton buffer, subsequently washed with PBS, and re-suspended in PBS buffer for flow cytometric analysis on the CytoFLEX LX Flow Cytometer. 50,000–100,000 cells were analyzed per sample per mouse using Beckman CytExpert Software.

Rong Q.X., Wang F., Guo Z.X., Hu Y., An S.N., Luo M., Zhang H., Wu S.C., Huang H.Q, & Fu L.W. (2021). GM-CSF mediates immune evasion via upregulation of PD-L1 expression in extranodal natural killer/T cell lymphoma. Molecular Cancer, 20, 80.

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