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Facsdiva version 8

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FACSDiva version 8.0.1 is a software application designed for the analysis and management of flow cytometry data. It provides a user interface for configuring and operating flow cytometry instruments, as well as tools for data acquisition, analysis, and reporting.

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Lab products found in correlation

Lsrfortessa, by BD (8 mentions) Lsrfortessa flow cytometer, by BD (6 mentions) Lsr 2 flow cytometer, by BD (4 mentions) Facscanto 2, by BD (4 mentions) Facscanto 2 cytometer, by BD (3 mentions) Lsrfortessa x 20, by BD (3 mentions) Zombie nir fixable viability stain, by BioLegend (3 mentions) Lsr 2, by BD (2 mentions) Ionomycin, by Merck Group (2 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Fixable live dead stain, by Thermo Fisher Scientific (2 mentions) Facsaria 3 sorter, by BD (2 mentions) Nylon mesh cell strainer snap caps, by STEMCELL (2 mentions) Tryple, by Thermo Fisher Scientific (2 mentions) Rna quick and easy kit, by Qiagen (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Lipofectamine l2000, by Thermo Fisher Scientific (2 mentions) Cytoxfix cytoperm, by BD (2 mentions) Live dead fixable yellow dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)

42 protocols using facsdiva version 8

1

Comprehensive Immune Cell Profiling

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The following fluorochrome-labeled anti-mouse mAbs were, unless otherwise stated, purchased from BD Biosciences: anti-CD11c (HL3), anti-IaIe (2G9), anti-CD3(145-2311), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-NK1.1 (PK136), anti-CD335 (29A1.4-eBiosciences), anti-CD19 (1D3), anti-CD11b (M1/70), anti-Gr1 (RB6-8C5), anti-F4/80 (BM8-Biolegend) and anti-Ly6C (AL-21). In addition, LIVE/DEAD® Fixable Yellow Dead Cell Stain Kit (Invitrogen) and diamidino-2-phenylindole (DAPI) (Invitrogen) were used for flow cytometric assessment. A minimum of 100,000 gated live cells were collected on a five-laser BD LSRFortessa (355, 405, 488, 532, and 640 nm; BD Biosciences). Data were analyzed using the FACSDiva Version 8.0.1 software (BD Biosciences).
Aydin E., Hallner A., Grauers Wiktorin H., Staffas A., Hellstrand K, & Martner A. (2018). NOX2 inhibition reduces oxidative stress and prolongs survival in murine KRAS-induced myeloproliferative disease. Oncogene, 38(9), 1534-1543.
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2

PKH26 Cell Membrane Labeling and Viability Analysis

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The cell membranes were labeled using the PKH26 Red Fluorescent Cell Linker kit (Sigma-Aldrich, Burlington, MA, USA, MIDI26-1KT) according to the manufacturer’s protocol. After 72 h in culture, the cells were stained with a viability dye and FLOW cytometry analysis was carried out using the BD FACSCanto II cytometer and analyzed using the FACSDiva Version 8.0.1 software (BD Biosciences, Franklin Lakes, NJ, USA).
El Naggar O., Doyle B., Mariner K, & Gilmour S.K. (2022). Difluoromethylornithine (DFMO) Enhances the Cytotoxicity of PARP Inhibition in Ovarian Cancer Cells. Medical Sciences, 10(2), 28.
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3

Phenotypic Profiling of Immune Cells

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Freshly isolated whole blood was collected from each study participant and was used to enumerate the percentage and subsets of γδ T cells, Vδ2 T cells, CD4 T cells, CD8 T cells and NK cells (the gating strategy is shown in Extended Data Fig. 10). Activated CD4 and CD8 T cells were defined by co-expression of HLA-DR. Samples were collected at enrolment and at days 2, 3, 7, 14 and 28 after each vaccination. In addition, whole blood was also collected immediately before CHMI and days 3, 7 and 14 after CHMI. In brief, 200 μl of whole blood from each participant was used to stain with a cocktail of conjugated monoclonal antibodies. The samples were washed, red blood cells lysed and washed again before acquisition on a BD LSRII flow cytometer equipped with a blue, red and violet laser, using BD FACSDiva version 8 software. All events in the tubes were acquired and analysed using FlowJo v.10.4.1 software.
Sohn M.H., Ursu S., Anderson J.R., Stenger V.A, & Carter C.S. (2000). The role of prefrontal cortex and posterior parietal cortex in task switching. Proceedings of the National Academy of Sciences of the United States of America, 97(24), 13448-13453.
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4

Phenotypic Profiling of Immune Cells

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Freshly isolated whole blood was collected from each study participant and was used to enumerate the percentage and subsets of γδ T cells, Vδ2 T cells, CD4 T cells, CD8 T cells and NK cells (the gating strategy is shown in Extended Data Fig. 10). Activated CD4 and CD8 T cells were defined by co-expression of HLA-DR. Samples were collected at enrolment and at days 2, 3, 7, 14 and 28 after each vaccination. In addition, whole blood was also collected immediately before CHMI and days 3, 7 and 14 after CHMI. In brief, 200 μl of whole blood from each participant was used to stain with a cocktail of conjugated monoclonal antibodies. The samples were washed, red blood cells lysed and washed again before acquisition on a BD LSRII flow cytometer equipped with a blue, red and violet laser, using BD FACSDiva version 8 software. All events in the tubes were acquired and analysed using FlowJo v.10.4.1 software.
Sohn M.H., Ursu S., Anderson J.R., Stenger V.A, & Carter C.S. (2000). The role of prefrontal cortex and posterior parietal cortex in task switching. Proceedings of the National Academy of Sciences of the United States of America, 97(24), 13448-13453.
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5

Flow Cytometry Analysis of Immune Cells

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Freshly isolated whole blood was collected from each study participant and was used to enumerate the percentage and subsets of γδ T cells, Vδ2 T cells, CD4 T cells, CD8 T cells and NK cells (the gating strategy is shown in Extended Data Fig. 10). Activated CD4 and CD8 T cells were defined by co-expression of HLA-DR. Samples were collected at enrolment and at days 2, 3, 7, 14 and 28 after each vaccination. In addition, whole blood was also collected immediately before CHMI and days 3, 7 and 14 after CHMI. In brief, 200 μl of whole blood from each participant was used to stain with a cocktail of conjugated monoclonal antibodies. The samples were washed, red blood cells lysed and washed again before acquisition on a BD LSRII flow cytometer equipped with a blue, red and violet laser, using BD FACSDiva version 8 software. All events in the tubes were acquired and analysed using FlowJo v.10.4.1 software.
Mwakingwe-Omari A., Healy S.A., Lane J., Cook D.M., Kalhori S., Wyatt C., Kolluri A., Marte-Salcedo O., Imeru A., Nason M., Ding L.K., Decederfelt H., Duan J., Neal J., Raiten J., Lee G., Hume J.C.C., Jeon J.E., Ikpeama I., Kc N., Chakravarty S., Murshedkar T., Church L.W.P., Manoj A., Gunasekera A., Anderson C., Murphy S.C., March S., Bhatia S.N., James E.R., Billingsley P.F., Sim B.K.L., Richie T.L., Zaidi I., Hoffman S.L, & Duffy P.E. (2021). Two chemoattenuated PfSPZ malaria vaccines induce sterile hepatic immunity. Nature, 595(7866).
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6

Competitive Fitness Assay with BTZ

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Next, 20 000 cells were infected at a multiplicity of infection of ∼1.2 to ensure 100% transduction efficiency with either virus particles of NLS-mCherry LacZ-sgRNA or NLS-BFP GOI-sgRNA. After transduction (96 hours), mCherry- and BFP-expressing cells were mixed 1:1 and plated with or without BTZ (4 nM) in a 12-well format. The cells were subcultured when near-confluency was reached, and BTZ containing medium was replaced every 3 days. Cells were imaged for BFP and mCherry signal the day of initial plating (t = 0) and on days 4, 8, 12, and 16. Data were acquired using the fluorescence-activated cell sorter (FACS) Fortessa cytofluorimeter and processed with FACSDiva version 8.0 software (BD Biosciences).
Findlay S., Nair R., Merrill R.A., Kaiser Z., Cajelot A., Aryanpour Z., Heath J., St-Louis C., Papadopoli D., Topisirovic I., St-Pierre J., Sebag M., Kesarwala A.H., Hulea L., Taylor E.B., Shanmugam M, & Orthwein A. (2023). The mitochondrial pyruvate carrier complex potentiates the efficacy of proteasome inhibitors in multiple myeloma. Blood Advances, 7(14), 3485-3500.
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7

Leukocyte Isolation and Phenotyping

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We lysed red blood cells in blood by red blood cell lysis buffer (eBioscience) and isolated leukocytes in brains by 37–70% Percoll (GE Healthcare) density gradient centrifugation. We counted cell numbers by a Coulter counter (Thermo Fisher). Cells were washed with buffer (PBS with 0.5% bovine serum abumin and 0.02% sodium azide) for three times and stained with antibodies listed in Supplementary Table 2. We used BD FACS LSRFortesa (12 fluorochromes, three-laser system, BD Biosciences) with FACS Diva version 8.0 software (BD Biosciences) to determine the phenotypes of leukocytes. We excluded dead cells by propidium iodide (Sigma, 20 µg ml−1 in PBS) positive staining and analyzed the data using Flowjo version 10 software (TreeStar).
Ren X., Hu H., Farooqi I, & Simpkins J.W. (2020). Blood substitution therapy rescues the brain of mice from ischemic damage. Nature Communications, 11, 4078.
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8

Annexin V Apoptosis Assay for Cisplatin

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Cells were seeded in 6-well plates at a density of 1.5×105 cells per well. Then cells were treated with different concentrations of cisplatin for 24 h, as indicated. Cells were harvested following cisplatin treatments, washed twice with cold PBS and centrifuged. The supernatants were discarded and the cells were resuspended in 1X Annexin-binding buffer. A total of 5 µl Annexin V-allophycocyanin (APC) or Annexin V-allophycocyanin (FITC) solution (BD Biosciences, Franklin Lakes, NJ, USA) was added to the cells at room temperature for 15 min, then 5 µl propidium iodide (PI) solution (BD Biosciences) was added. The ratio of apoptotic cells (% Annexin V-APC or Annexin V-FITC positive cells per total) was measured by flow cytometry (BD FACSDiva version 8.0.1; BD Biosciences).
Li C., Cai J., Ge F, & Wang G. (2018). TGM2 knockdown reverses cisplatin chemoresistance in osteosarcoma. International Journal of Molecular Medicine, 42(4), 1799-1808.
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9

Apoptosis and Cytokine Analysis of hAMSCs

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In order to analyze cell apoptosis/necrosis in hAMSCs grown in serum-free culture conditions, annexin-V/7-AAD assay kits (BD Biosciences, USA) for cell staining were used following the manufacturer's instructions. Their fluorescences were detected using a BD FACSCanto II instrument, and the data were analyzed with BDFACSDiva version 8.0.1 (BD Biosciences, USA). The levels of different cytokines and growth factors in each conditioned medium were determined using magnetic bead technology from Luminex™ with the ProcartaPlex Human Cytokine Chemokine Growth Factor (Affymetrix, USA) according to the manufacturer's instructions. The concentration of each factor was calculated from standard curves.
Lo Nigro A., Gallo A., Bulati M., Vitale G., Paini D.S., Pampalone M., Galvagno D., Conaldi P.G, & Miceli V. (2021). Amnion-Derived Mesenchymal Stromal/Stem Cell Paracrine Signals Potentiate Human Liver Organoid Differentiation: Translational Implications for Liver Regeneration. Frontiers in Medicine, 8, 746298.
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10

Antibody Staining and Flow Cytometry

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Antibodies and dilutions used in this study are listed in Supplementary Table 2. Antibody staining was performed in the presence of purified CD16/CD32 antibody (Mouse BD Fc-Block™, clone: 2.4G2, #553141, BD Biosciences) at a concentration of 1 µg per 1 × 106 cells. If required, cells were stained after RBC‐lysis with 1.66% NH4Cl w/v. Live/Dead cell discrimination was performed by the addition of Zombie Green™ Fixable Viability Dye (#423111, BioLegend). Flow cytometry was performed using BD LSRFortessa™ or BD FACSLyric™ instruments. Data were acquired using BD FACSDiva™ Version 8.0.1 and BD FACSuite™ 1.2.1 (BD Biosciences). Data were analyzed using FlowJo™ v10.7.1 software (BD Biosciences).
Koerner J., Horvath D., Herrmann V.L., MacKerracher A., Gander B., Yagita H., Rohayem J, & Groettrup M. (2021). PLGA-particle vaccine carrying TLR3/RIG-I ligand Riboxxim synergizes with immune checkpoint blockade for effective anti-cancer immunotherapy. Nature Communications, 12, 2935.
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