Facsymphony a5 cell analyzer

Manufactured by BD

Sourced in United States

The BD FACSymphony A5 Cell Analyzer is a flow cytometry instrument designed for high-parameter, high-throughput analysis of cellular samples. It is capable of detecting and analyzing up to 20 parameters simultaneously, allowing for in-depth characterization of complex cell populations.

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Lab products found in correlation

Zombie uv fixable viability dye kit, by BioLegend (6 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Propidium iodide, by Thermo Fisher Scientific (3 mentions) Aquavivid, by Thermo Fisher Scientific (2 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Rainbow beads, by Spherotech (2 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions) Apc anti human cd57 antibody, by BioLegend (2 mentions) Fitc anti human cd56 ncam antibody, by BioLegend (2 mentions) Optibuild buv395 anti human cd8, by BD (2 mentions) Brilliant violet 421 anti human cd159a nkg2a antibody, by BioLegend (2 mentions) Pe anti human cd159c nkg2c antibody, by BioLegend (2 mentions) Apc cyanine7 anti human cd127 il 7rα antibody, by BioLegend (2 mentions) Brilliant violet 785 anti mouse human klrg1 mafa antibody, by BioLegend (2 mentions) Pe cyanine7 anti human cd16 antibody, by BioLegend (2 mentions) Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions) Anti cd8 v450, by BD (1 mentions) Brefeldin a, by Thermo Fisher Scientific (1 mentions) Flojo software, by Tree Star (1 mentions)

39 protocols using facsymphony a5 cell analyzer

Evaluating Transfection Efficiency and Cell Cycle

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To assess transfection efficiency, cells were plated in 6-well plates and transfected with plasmids. Cells were collected 24 hours post-transfection and stained with cell viability dye FVD-eFluor-506 (Thermo Fisher Scientific) at 4°C for 30 min, then subjected to flow cytometry analysis using a BD FACSymphony A5 Cell Analyzer (Indianapolis, IN). The GFP-positive live cell population was calculated by FlowJo (FlowJo LLC, Ashland, OR). For cell cycle analysis, cells were collected and washed twice with PBS, then resuspended in 1 mL PBS and fixed by adding 4 mL ice-cold absolute ethanol at -20°C overnight. After fixation, cells were washed and counter-stained with a PBS-buffered solution containing 0.1% Triton X-100, 3 µM propidium iodide (PI, Thermo Fisher Scientific), and 0.5 mg/mL RNase A (Sigma Aldrich) at 4°C for 30 min. Cell cycle distribution was examined using a BD FACSymphony A5 Cell Analyzer, and the proportion of cells in the G0−G1, S, and G2−M phases was determined using FlowJo software. All experiments were performed in triplicate.

Geng H., Subramanian S., Wu L., Bu H.F., Wang X., Du C., De Plaen I.G, & Tan X.D. (2021). SARS-CoV-2 ORF8 Forms Intracellular Aggregates and Inhibits IFNγ-Induced Antiviral Gene Expression in Human Lung Epithelial Cells. Frontiers in Immunology, 12, 679482.

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Intracellular Cytokine Staining of CD8+ T Cells

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CD8⁺ T cells were sorted and cultured as described in Sections 2.2 and 2.3. For the intracellular measurement of interferon‐γ (IFN‐γ), granzyme B (GZMB) and perforin‐1 (PRF‐1), the CD8⁺ T cell subsets were incubated with 3 μg/ml brefeldin A (Thermo Fisher Scientific, Waltham, MA) for 6 h. The harvested cells were then stained with anti‐CD8‐V450 (for details see above), anti‐IFN‐γ‐Alexa Fluor 488 (clone: B27, RRID:AB_396827), anti‐granzyme B‐Alexa Fluor 647 (clone: GB11, RRID:AB_10897997) and anti‐perforin‐1‐PE‐CF594 (clone: δG9 RRID:AB_2738410), all obtained from BD Biosciences (San Diego, CA) using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit (BD Biosciences San Diego, CA) according to the manufacturer's instructions. All antibodies were titrated to determine the optimal concentration for use. The stained samples were acquired with a FACSymphony™ A5 Cell Analyzer (BD Biosciences, Heidelberg, Germany). The FMO stainings were used for gating. The data were analysed using FlowJo software V10.7.1 (BD Life Sciences).

Burkard T., Herrero San Juan M., Dreis C., Kiprina A., Namgaladze D., Siebenbrodt K., Luger S., Foerch C., Pfeilschifter J.M., Weigert A, & Radeke H.H. (2022). Differential expression of CD8 defines phenotypically distinct cytotoxic T cells in cancer and multiple sclerosis. Clinical and Translational Medicine, 12(12), e1068.

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Quantifying Surface CD8-CI-MPR Expression

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To assess the levels of surface CD8-CI-MPR, unfixed cells were incubated for 30 min at 4 °C with mouse anti-CD8 antibody (9 (link), 59 (link)) followed by incubation for 30 min at 4 °C with F(ab')2-Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody Alexa Fluor 647 (Cat. A21237; Thermo Scientific) in PBS supplemented with 1% BSA, and then fixed with PFA 1% in PBS + 1% BSA and subjected to flow cytometry analysis. Data were acquired using the levels of Alexa 647 fluorescence in cells from a BD LSRFortessa flow cytometer (BD Biosciences) and FACSymphony A5 Cell Analyzer (BD Biosciences) at the Ribeirão Preto Center for Cell-Based Therapy/Hemotherapy. The FloJo software (Tree Star) was used for data analyses.

Tavares L.A., Rodrigues R.L., Santos da Costa C., Nascimento J.A., Vargas de Carvalho J., Nogueira de Carvalho A., Mardones G.A, & daSilva L.L. (2024). AP-1γ2 is an adaptor protein 1 variant required for endosome-to-Golgi trafficking of the mannose-6-P receptor (CI-MPR) and ATP7B copper transporter. The Journal of Biological Chemistry, 300(3), 105700.

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Comprehensive PBMC Surface Marker Analysis

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PBMCs were resuspended at 4x10⁶ cells/mL in RPMI (Gibco by Life Technologies) supplemented with penicillin/streptomycin (Gibco by Life Technologies), 10% heat inactivated FCS and incubated at 37°C, 5% CO₂ for 2hrs in the presence of fluorescently labeled CCR10 antibody.
For surface stain, PBMCs were first stained for viability dye (Aquavivid, Thermofisher, 20min, 4°C) next with a mix containing a brilliant stain buffer (BD Biosciences), the surface markers for B cells detection (CD19, CD20, CD21, IgM and IgD), B cells memory phenotype (CD24, CD27, IgG and IgA), plasmablasts and plasma cells (CD38 and CD138) phenotypes, T-cells and monocytes exclusion (CD3, CD56, CD14, and CD16) (30min, 4°C) (see Table S2 for antibodies), as well as fluorescently-labeled probes for RBD⁺ B cells detection targeting two different epitopes of the RBD (RBD1-AF488 and RBD2 AF594). Omicron BA.1-RBD peptide (Accrobiosystem) was labeled, and the Omicron BA.1-RBD probe was also added into the mix where appropriate (RBD Omicron BA.1 AF647). Cells were fixed with 1% paraformaldehyde (Sigma-Aldritch) for 15 min at room temperature before filtration for acquisition on a FACSymphony A5 Cell Analyzer (BD Biosciences) and analyzed using FlowJo (BD, v10.6.2).

Sannier G., Nicolas A., Dubé M., Marchitto L., Nayrac M., Tastet O., Chatterjee D., Tauzin A., Lima-Barbosa R., Laporte M., Cloutier R., Sreng Flores A.M., Boutin M., Gong S.Y., Benlarbi M., Ding S., Bourassa C., Gendron-Lepage G., Medjahed H., Goyette G., Brassard N., Delgado G.G., Niessl J., Gokool L., Morrisseau C., Arlotto P., Rios N., Tremblay C., Martel-Laferrière V., Prat A., Bélair J., Beaubien-Souligny W., Goupil R., Nadeau-Fredette A.C., Lamarche C., Finzi A., Suri R.S, & Kaufmann D.E. (2023). A third SARS-CoV-2 mRNA vaccine dose in people receiving hemodialysis overcomes B cell defects but elicits a skewed CD4+ T cell profile. Cell Reports Medicine, 4(3), 100955.

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Annexin-V-Alexa Fluor 568 Apoptosis Assay

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A total of 100,000 cells were seeded per well in 6-well plates and left seated for 24 h. Cells were treated with ND-Nic for 48 h, respectively. According to the manufacturer’s protocol, cells were harvested, pelleted, and resuspended in 130 µL of freshly prepared Annexin-V-Fluos labelling solution comprising Annexin-V-Alexa Fluor 568 (Roche Diagnostics USA 03 703 126 001) and HEPES incubation buffer (Roche Diagnostics USA 11 858 777 001) at a 1:50 (v/v) ratio. After 20 min of incubation in the dark at room temperature, 300 µL of 1× PBS was added to each sample before proceeding to flow cytometry analysis (BD Biosciences FACSymphony™ A5 Cell Analyzer) using a 610/30 BP filter.

Ngai T.W., Elfar G.A., Yeo P., Phua N., Hor J.H., Chen S., Ho Y.S, & Cheok C.F. (2021). Nitro-Deficient Niclosamide Confers Reduced Genotoxicity and Retains Mitochondrial Uncoupling Activity for Cancer Therapy. International Journal of Molecular Sciences, 22(19), 10420.

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Multi-Marker Flow Cytometry Analysis

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Peripheral blood was collected and treated with Gey’s solution to remove red blood cells. Then, the remaining cells were resuspended in phosphate-buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS). These cells were stained with a combination of fluorescence-conjugated antibodies. Antibodies, including APC-conjugated anti-B220 (553092) and anti-CD4 (553051), PE-Cy7-conjugated anti-Mac-1 (561098), PE-conjugated anti-Gr-1 (561084), BUV805-conjugated anti-CD8 (612898), and FITC-conjugated anti-CD25 (553072), were purchased from BD Biosciences. Alexa Fluor 700-conjugated anti-CD3 (100216) was purchased from Biolegend. The samples were analyzed using the FACSymphony™ A5 cell analyzer (BD Biosciences, Franklin Lake, NJ, USA), and the data were analyzed using FlowJo v10.8 software.

Wei J., Liu C., He X., Abbas B., Chen Q., Li Z, & Feng Z. (2023). Generation and Characterization of Recombinant Pseudorabies Virus Delivering African Swine Fever Virus CD2v and p54. International Journal of Molecular Sciences, 25(1), 335.

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Stimulation and Intracellular Cytokine Staining of PBMCs

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PBMCs were resuspended at 10x10⁶ cells/mL RPMI (Gibco by Life Technologies) supplemented with penicillin/streptomycin (Gibco by Life Technologies), 10% heat inactivated FCS, and incubated at 37°C, 5% CO₂. After a rest of 2hrs, cells were stimulated with 0.5 μg/mL staphylococcal enterotoxin B (SEB) or 0.5 μg/mL of overlapping peptide pools for Wuhan-1 or Omicron BA.1 variants SARS-CoV-2 Spike (JPT) for 6 hrs at 37°C, 5% CO₂. An unstimulated condition with 0.4μL of DMSO served as a negative control. Brefeldin A (BD Biosciences), Monensin-1 (BD Biosciences), and a fluorescently labeled CD107a antibody were added for the remaining 5hrs.
Cells were stained for viability dye (Aquavivid, Thermofisher, 20min, 4°C), surface markers (30min, 4°C), and intracellularly for cytokines (30min, room temperature) using the IC Fixation/Permeabilization kit (eBioscience) (see Table S4 for antibodies) and filtrated before acquisition on the flow cytometer (FACSymphony A5 Cell Analyzer, BD Biosciences) and analyzed using FlowJo (BD, v10.6.2).

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Detecting SARS-CoV-2-Specific B Cells

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To detect SARS-CoV-2-specific B cells, we conjugated recombinant RBD proteins with Alexa Fluor 488 or Alexa Fluor 594 (Thermo Fisher Scientific) according to the manufacturer’s protocol. Approximately 2 × 10⁶ frozen PBMCs from SARS-CoV-2 naive and prior infection donors were prepared in Falcon® 5ml-round bottom polystyrene tubes at a final concentration of 4 × 10⁶ cells/mL in RPMI 1640 medium (GIBCO) supplemented with 10% of fetal bovine serum (Seradigm), Penicillin- Streptomycin (GIBCO) and HEPES (GIBCO). After a rest of 2 h at 37°C and 5% CO₂, cells were stained using Aquavivid viability marker (Biosciences) in DPBS (GIBCO) at 4°C for 20 min. The detection of SARS-CoV-2-antigen specific B cells was done by adding the RBD probes to the antibody cocktail (IgM BUV737, CD24 BUV805, IgG BV421, CD3 BV480, CD56 BV480, CD14 BV480, CD16 BV480, CD20 BV711, CD21 BV786, HLA DR BB700, CD27 APC R700; CD19 BV650, CD38 BB790, CD138 BUV661, CCR10 BUV395, IgD BUV563 and IgA PE). Staining was performed at 4°C for 30 min and cells were fixed using 1% paraformaldehyde (Sigma-Aldrich) at 4°C for 15 min. Stained PBMC samples were acquired on FACSymphony A5 Cell Analyzer (BD Biosciences) and analyzed using FlowJo v10.7.1 software.

Tauzin A., Gong S.Y., Beaudoin-Bussières G., Vézina D., Gasser R., Nault L., Marchitto L., Benlarbi M., Chatterjee D., Nayrac M., Laumaea A., Prévost J., Boutin M., Sannier G., Nicolas A., Bourassa C., Gendron-Lepage G., Medjahed H., Goyette G., Bo Y., Perreault J., Gokool L., Morrisseau C., Arlotto P., Bazin R., Dubé M., De Serres G., Brousseau N., Richard J., Rovito R., Côté M., Tremblay C., Marchetti G.C., Duerr R., Martel-Laferrière V., Kaufmann D.E, & Finzi A. (2021). Strong humoral immune responses against SARS-CoV-2 Spike after BNT162b2 mRNA vaccination with a 16-week interval between doses. Cell Host & Microbe, 30(1), 97-109.e5.

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Neutralization Assay of Inactivated Sera

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Neutralization of irradiated and heat-inactivated serum samples were assessed in Vero UNC cells. Neutralization assays with VSV-TAFV-GFP were performed as previously described [16 (link)]. Samples were run on the FACSymphony A5 Cell Analyzer (BD Biosciences, Mississauga, ON, Canada) where the GFP-positive cell count was measured and data analysed on FlowJo V10.

Fletcher P., O’Donnell K.L., Doratt B.M., Malherbe D.C., Clancy C.S., Rhoderick J.F., Feldmann F., Hanley P.W., Ksiazek T.G., Geisbert T.W., Messaoudi I, & Marzi A. (2023). Single-dose VSV-based vaccine protects cynomolgus macaques from disease after Taï Forest virus infection. Emerging Microbes & Infections, 12(2), 2239950.

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SARS-CoV-2-specific T cell activation assay

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HLA-A*02:01 SARS-CoV-2-spheromers were conjugated with PE and the following spike protein epitopes (ORF1ab3467, ORF1ab4032, ORF1ab4725, S976, S983) were used at 1:10 in FACS buffer⁶⁰. Surface staining antibodies were added at 1:100 and live/dead Fixable Near IR 780 staining (ThermoFisher, L34992) were added at 1:1000. Cells were then fixed with Cytofix (BD, 554714) and permeabilized with 0.5% saponin and stained with the following antibodies at 1:100 dilution: anti-human CD40L, anti-human OX40, anti-human CD137 (antibody details in Supplementary Table 4). Data were acquired with BD FACSymphony^™ A5 Cell Analyzer and analyzed with FlowJo. HLA-A2-expression on organoids used for this analysis was confirmed by FACS.

Choi S.S., van Unen V., Zhang H., Rustagi A., Alwahabi S.A., Santos A.J., Chan J.E., Lam B., Solis D., Mah J., Röltgen K., Trope W., Guh-Siesel A., Lin Z., Beck A., Edwards C., Mallajosyula V., Martin B.A., Dunn J.C., Shrager J., Baric R.A., Pinsky B., Boyd S.D., Blish C.A., Davis M.M, & Kuo C.J. (2023). Organoid modeling of lung-resident immune responses to SARS-CoV-2 infection. Research Square.

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