Facsdiva software v8

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FACSDiva software v8.0 is a cytometry data acquisition and analysis software developed by BD Biosciences. The software's core function is to provide users with a graphical user interface to control flow cytometry instrumentation, acquire and analyze cell population data.

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BD FACSDiva v8.0.1 (158 citations) FACSDiva software (5426 citations) FACSDiva (1830 citations)

106 protocols using facsdiva software v8

Flow Cytometry Analysis of PBMC and CSF

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Cells were always analyzed within 1 h of staining. Mean autofluorescence values were set using appropriate negative isotype controls. Data analysis was performed using FACSDiva Software V.8.0 (BD Biosciences). A gate including lymphocytes and monocytes and excluding debris and apoptotic cells was established; a minimum amount of 30,000 events for PBMC samples and 500 events for CSF cells were analyzed.

Malhotra S., Villar L.M., Costa C., Midaglia L., Cubedo M., Medina S., Fissolo N., Río J., Castilló J., Álvarez-Cermeño J.C., Sánchez A., Montalban X, & Comabella M. (2018). Circulating EZH2-positive T cells are decreased in multiple sclerosis patients. Journal of Neuroinflammation, 15, 296.

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Multiparametric Flow Cytometry Analysis of Immune Cells

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Multiparametric flow cytometry was used for phenotypic and functional analysis of PBMC, IHL and TILs. Cells were stained with a fixable Live/Dead dye (Invitrogen) before incubation with saturating concentrations of surface mAbs diluted in 50% Brilliant Stain buffer (BD Biosciences) and 50% PBS (Invitrogen) for 15 min at 4 °C. Cells were fixed and permeabilized for further functional assessment with Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s instructions. Cells were incubated with saturated concentrations of mAbs diluted in a 0.1% saponin-based buffer (Sigma-Aldrich) for 20 min at 4 °C for the detection of intracellular proteins. All samples were acquired on Fortessa X20 flow cytometer using FACSDiva software v8.0 (BD biosciences) and analysed using FlowJo v.9 (Tree Star; BD Biosciences). Full details on monoclonal antibodies used can be found in Supplementary Table 5.

Zakeri N., Hall A., Swadling L., Pallett L.J., Schmidt N.M., Diniz M.O., Kucykowicz S., Amin O.E., Gander A., Pinzani M., Davidson B.R., Quaglia A, & Maini M.K. (2022). Characterisation and induction of tissue-resident gamma delta T-cells to target hepatocellular carcinoma. Nature Communications, 13, 1372.

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Memory T Cell-Epithelial Interaction Assay

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Primary human keratinocytes and fibroblasts were cultured to confluence in 96-well flat-bottomed plates. T cells from the three sorted memory subtypes were stimulated with αCD3/CD2/CD28 T cell activation beads (Miltenyi Biotec, San Diego, CA) at a 1:10 ratio for 24 hr. Stimulated memory T cells from each subtype were added to designated wells of confluent layers of keratinocytes, fibroblasts, or tissue culture wells without keratinocytes or fibroblasts (plastic) in Iscove’s Modified Dulbecco’s Medium supplemented with 10% FBS and L-glutamine. At indicated time points, T cells were collected and immunostained with directly conjugated anti-human monoclonal antibodies from BioLegend and Life Technologies. Acquisition of flow cytometry samples was performed on a FACS Canto instrument and data were analyzed using FACS Diva software V8.0 (BD Biosciences).

Matos T.R., Gehad A., Teague J.E., Dyring-Andersen B., Benezeder T., Dowlatshahi M., Crouch J., Watanabe Y., O’Malley J.T., Kupper T.S., Yang C., Watanabe R, & Clark R.A. (2022). Central memory T cells are the most effective precursors of resident memory T cells in human skin. Science immunology, 7(70), eabn1889.

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Plasmid Inheritance Stability Assay

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M. extorquens carrying pK18mob2-repABC derivatives were grown until OD₆₀₀=1.0-1.5 in selective medium. Cell suspensions were diluted every 24 hours to an OD₆₀₀≈0.02 in non-selective medium. Dilution series were plated on agar with and without kanamycin. Antibiotic resistance was correlated with the presence of the assessed plasmid. The inheritance stability of selected repABC replicons expressing mCherry as mini-chromosomes was assessed via flow cytometry on a BD LSRFortessaTM SORP flow-cytometer (BD Biosciences, NJ, USA). Fluorescence was detected using a 561 nm laser at 100 mW and a 610/20 bandpass filter^{30 (link), 31 (link)}. Forward and side scatter values were monitored using a 488 nm laser at 100 mW. The acquired data was analyzed using FACSDiva™ software v8.0 (BD Biosciences). The percentage of fluorescent and non-fluorescent cells was determined from 30,000 gated events each.

Döhlemann J., Wagner M., Happel C., Carrillo M., Sobetzko P., Erb T.J., Thanbichler M, & Becker A. (2017). A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti. ACS Synthetic Biology, 6(6), 968-984.

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Murine Microglia Phagocytosis Assay

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Murine primary microglia prepared as described above were grown in DMEM containing 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin. For phagocytosis assays, the cells were seeded at 8 × 10⁵cells/well into 6-well plates. After 24 h, the medium was replaced with DMEM containing 0.5% FBS, protein-coupled microspheres (10 beads/cell ratio) were added to each well, and the plates were incubated for 30 min at 37 °C in the presence or absence of an anti-CD44 (1 μg/mL; IM7) or anti-αv integrin neutralizing antibody (1 μg/mL; clone RMV-7, 550024, BD). The cells were then washed 3 times with cold PBS, collected in FACS buffer (PBS containing 2% FBS) and examined using flow cytometry in a LSR II Flow Cytometer running FACSDiva Software v8.0 (BD). A total of 10,000 events were collected from each sample.

Morisaki Y., Niikura M., Watanabe M., Onishi K., Tanabe S., Moriwaki Y., Okuda T., Ohara S., Murayama S., Takao M., Uchida S., Yamanaka K, & Misawa H. (2016). Selective Expression of Osteopontin in ALS-resistant Motor Neurons is a Critical Determinant of Late Phase Neurodegeneration Mediated by Matrix Metalloproteinase-9. Scientific Reports, 6, 27354.

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Flow Cytometric Analysis of Dendritic Cell Activation

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For flow cytometric analysis, 5 × 10⁵ moDCs/mL were treated with 10 µg/mL BPE and 2.1 µg/mL Bet v 1 + 1.0 ng/mL LPS respectively (Fig. S1e). After incubation for 24 h, cells were washed with ice-cold DPBS and stained in 100 μl DPBS containing fluorescent-labelled antibodies for 30 min under protection from light. After extensive washing, staining was fixed by adding 4% (v/v) paraformaldehyde (PFA, Sigma-Aldrich) for 10 min. Then, cells were admitted into 200 µL DPBS containing 3 mM EDTA. The median fluorescence intensity (MFI) of 1 × 10⁴ cells was recorded for every sample using the FACS Canto™ II flow cytometer operated with FACS Diva Software v8.0 (BD Biosciences, Vienna, Austria). Data analysis was performed using the FlowJo^® Software v10.3 (FlowJo LLC, Ashland, Oregon, USA).
EBioscience™ fixable Viability Dye eFluor® 506, anti-human CD3 eFluor® 506 (clone: UCHT1), anti-human CD19 eFluor® 506 (HIB19), anti-human CD40 FITC (5C3), anti-human CD86 PE (IT2.2), and anti-human HLA-DR APC (LN3) were purchased from Thermo Fisher Scientific. Brilliant Violet 421™ anti-human CD1a (Hl149) was obtained from BioLegend (San Diego, CA, USA). APC-H7 mouse anti-human CD80 (L307.4) and PE-Cy™7 mouse anti-human CD83 (HB15e) were purchased from BD Biosciences.

Strasser L., Dang H.H., Schwarz H., Asam C., Ferreira F., Horejs-Hoeck J, & Huber C.G. (2017). Unbiased Quantitative Proteomics Reveals a Crucial Role of the Allergen Context for the Activation of Human Dendritic Cells. Scientific Reports, 7, 16638.

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Immunophenotyping of Frozen PBMCs

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Frozen PBMC were thawed and washed twice with PBA buffer (phosphate buffer saline, 1% bovine serum albumin, 10 mM NaN3). Cells were then stained with the antibody combination reported in Supplementary Table 4, in a final volume of 100 uL PBA for 30 min at 4 °C. Cells were washed 2x in PBA, fixed with 2% paraformaldehyde (PFA), and acquired on a FACSymphony flow cytometer (BD Biosciences) with the FACSDiva software v8.0 (BD Biosciences). Analysis was carried out with the FlowJo v10.4 software.

Claireaux M., Robinot R., Kervevan J., Patgaonkar M., Staropoli I., Brelot A., Nouël A., Gellenoncourt S., Tang X., Héry M., Volant S., Perthame E., Avettand-Fenoël V., Buchrieser J., Cokelaer T., Bouchier C., Ma L., Boufassa F., Hendou S., Libri V., Hasan M., Zucman D., de Truchis P., Schwartz O., Lambotte O, & Chakrabarti L.A. (2022). Low CCR5 expression protects HIV-specific CD4+ T cells of elite controllers from viral entry. Nature Communications, 13, 521.

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Multiparameter Analysis of Cell Death and T Cell Cytokines

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For cell death analysis, cells were treated, collected, and initially stained with specific antibodies, then resuspended in PBS containing 1 μg/ml Propidium Iodide (PI) or 7-Aminoactinomycin D (7-AAD) for 5 minutes, and directly run on a flow cytometer. For cells expressing intracellular fluorescence proteins, cells were resuspended in PBS containing 1 μl LIVE/DEAD® Fixable Blue Dead Cell Stain (Thermo Fisher Scientific) for 20 minutes, then analyzed.
To quantify T cells and effector T cell cytokine expression, single-cell suspensions were prepared from fresh tumor tissues. T cells were enriched by density gradient centrifugation. For cytokine staining, T cells were incubated in culture medium containing PMA (5 ng/ml), Ionomycin (500 ng/ml), Brefeldin A (1: 1000) and Monensin (1: 1000) at 37°C for 4 hours. Anti-CD45 (30-F11), anti-CD90 (53–2.1), anti-CD4 (RM4–5), and anti-CD8 (53–6.7) were added for 20 minutes for surface staining. The cells were then washed and resuspended in 1 ml of freshly prepared Fix/Perm solution (BD Biosciences) at 4°C for overnight. After being washed with Perm/Wash buffer (BD Biosciences), the cells were stained with anti-TNFα (MP6-XT22) and anti-IFNγ (XMG1.2) for 30 minutes, washed, and fixed in 4% formaldehyde (Sigma Aldrich). All samples were read on an LSR II cytometer and analyzed with FACS DIVA software v. 8.0 (BD Biosciences).

Wang W., Green M., Choi J.E., Gijón M., Kennedy P.D., Johnson J.K., Liao P., Lang X., Kryczek I., Sell A., Xia H., Zhou J., Li G., Li J., Li W., Wei S., Vatan L., Zhang H., Szeliga W., Gu W., Liu R., Lawrence T., Lamb C., Tanno Y., Cieslik M., Stone E., Georgiou G., Chan T.A., Chinnaiyan A, & Zou W. (2019). CD8+ T cells regulate tumor ferroptosis during cancer immunotherapy. Nature, 569(7755), 270-274.

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Isolation and FACS of Liver Nonparenchymal Cells

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Liver nonparenchymal cells were isolated from mouse liver as previously described.⁽²¹⁾ Subsequently, antibodies (Brilliant Violet 421‐CD31 [BioLegend, San Diego, CA] and PE‐PDGFRβ [BioLegend]) and their isotype‐matched negative control antibodies were added to the cell suspension. After 30 minutes of incubation in the dark, the cells were washed with phosphate‐buffered saline and subjected to fluorescence‐activated cell sorting (FACS), which was performed on FACSAria IIIu and analyzed with FACSDiva software v8.0 (BD Biosciences, Franklin Lakes, NJ).

Yang L., Yue W., Zhang H., Zhang Z., Xue R., Dong C., Liu F., Chang N., Yang L, & Li L. (2022). Dual Targeting of Angipoietin‐1 and von Willebrand Factor by microRNA‐671‐5p Attenuates Liver Angiogenesis and Fibrosis. Hepatology Communications, 6(6), 1425-1442.

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Multiparametric Flow Cytometry Analysis

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To study surface antigens, cells were stained with the respective monoclonal antibodies during 30 min at 4°C in the dark, washed with saline and analyzed in a FACSCanto II flow cytometer (BD Biosciences), as described before (12 (link)).
For intracytoplasmic cytokine detection, cells were stimulated with phorbol-12-myristate-13-acetate (PMA, Merck) and Ionomycin (Merck), in presence of Brefeldin A (GolgiPlug, BD Biosciences) and Monensin (GolgiStop, BD Biosciences), and then incubated 4 h at 37°C in 5% CO2 atmosphere. For the analysis of IL-10 producing B cells, PBMC were incubated prior to stimulation with CpG oligonucleotide (InvivoGen) for 20 h. After incubation, surface markers were stained, then the cellular membrane permeabilized with Cytofix/Cytoperm Kit (BD Biosciences), and incubated with intracellular antibodies, following the same protocol previously described (12 (link)).
Cells were analyzed using FACSDiva Software V.8.0 (BD Biosciences). A minimum amount of 5x10 (4 (link)) events were acquired. Gating strategy is defined in Figure 1.

Walo-Delgado P.E., Monreal E., Medina S., Quintana E., Sainz de la Maza S., Fernández-Velasco J.I., Lapuente P., Comabella M., Ramió-Torrentà L., Montalban X., Midaglia L., Villarrubia N., Carrasco-Sayalero A., Rodríguez-Martín E., Roldán E., Meca-Lallana J., Alvarez-Lafuente R., Masjuan J., Costa-Frossard L, & Villar L.M. (2021). Role of B Cell Profile for Predicting Secondary Autoimmunity in Patients Treated With Alemtuzumab. Frontiers in Immunology, 12, 760546.

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