Diva acquisition software

Manufactured by BD

Sourced in United States

Diva acquisition software is a data acquisition and analysis tool developed by BD for use with their laboratory instruments. The software is designed to capture and process data from various BD instruments, enabling users to monitor and analyze their experimental results.

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Lab products found in correlation

Versacomp antibody capture beads, by Beckman Coulter (4 mentions) Flojo analysis software, by Tree Star (2 mentions) Facscanto 2 flow cytometer, by BD (2 mentions) Fc block anti cd16 32, by Cytek Biosciences (2 mentions) Lsr 2 flow cytometer, by BD (2 mentions) Lsrii flow cytometer, by BD (1 mentions) Percp cy5, by BioLegend (1 mentions) Apc fire 750, by BioLegend (1 mentions) Bv605, by BioLegend (1 mentions) Facs lsrfortessa, by BD (1 mentions) Bv421, by BioLegend (1 mentions) Staining buffer, by BioLegend (1 mentions) Lsrii cytometer, by BD (1 mentions) Tryple express, by Thermo Fisher Scientific (1 mentions) Neural tissue dissociation kit, by Miltenyi Biotec (1 mentions) Tq prep, by Beckman Coulter (1 mentions) Gentlemacs dissociator, by Miltenyi Biotec (1 mentions) Clodronate liposomes, by Merck Group (1 mentions) Cd68 pe, by BioLegend (1 mentions) Ly6c apc cy7, by BioLegend (1 mentions)

9 protocols using diva acquisition software

Phenotypic Switching Analysis via Flow Cytometry

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The analysis of phenotypic switching was performed as described previously (2 (link)), using BD FACSCanto™ II Flow Cytometer (Becton, Dickinson and Company, USA) with Diva™ acquisition software (Becton Dickinson), FloJo™ analysis software (Tree Star, Inc. USA) and Prism™ (GraphPad). All cultures were subjected to arbitrary gating, which encompassed approximately 70% of the most typical events (Supplementary Figure S1).

Satory D., Gordon A.J., Wang M., Halliday J.A., Golding I, & Herman C. (2015). DksA involvement in transcription fidelity buffers stochastic epigenetic change. Nucleic Acids Research, 43(21), 10190-10199.

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Measuring Lac Operon Induction by Flow Cytometry

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To determine the percentage of cells that were induced for lac operon expression (ON cells), 10 μl of culture was diluted into 300 μl filtered minimal A salts plus MgSO₄ (1 mM) and subjected to flow cytometry analysis with GFP fluorescence measured in a BD FACSCanto II Flow Cytometer (Becton, Dickinson and Company, USA) with Diva acquisition software (Becton Dickinson) and FloJo analysis software (Tree Star, Inc. USA). To monitor fluorescent cells in a culture we used a narrow gating for forward and side scattering so that the most represented cell population was evaluated. For each independent culture 10 000 cells were interrogated, as previously described (6 (link)).

Gordon A.J., Satory D., Wang M., Halliday J.A., Golding I, & Herman C. (2014). Removal of 8-oxo-GTP by MutT hydrolase is not a major contributor to transcriptional fidelity. Nucleic Acids Research, 42(19), 12015-12026.

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Microglia Immunophenotyping in Neurological Injury

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Animals were euthanized at either 7 or 30 days after injury. Upon sacrifice, brains were harvested and processed as described above.
The isolated brain cells were immunophenotyped using a modified version of our previously published microglia immunophenotyping strategy using the following cell surface antibodies: anti‐CD45, anti‐CD11bc, anti‐P2Y12, anti‐CD32, anti‐RT1B, and anti‐CD163.¹⁹ Briefly, the cells were stained with the Ghost live/dead reagent and incubated for 15 minutes at RT in the dark. The cells were then washed with 1 mL of staining buffer and centrifuged at 400g for 5 minutes. The supernatant was removed, and the cells were then stained with the antibody mix mentioned in Table 1 and incubated for 30 minutes at RT in the dark. Cells were washed again with staining buffer and centrifuged at 400g for 5 minutes. The supernatant was removed, and the secondary antibody mix was added. The cells were incubated for 20 minutes at RT in the dark. The cells were then gently vortexed, and 25 μL of counting control beads (Cyto‐Cal) were added. Data were acquired with LSR‐II flow cytometer with Diva acquisition software (BD Biosciences). Fluorescence spillover compensation values were generated using VersaComp Antibody Capture Beads (Beckman Coulter).

Caplan H.W., Prabhakara K.S., Kumar A., Toledano‐Furman N.E., Martin C., Carrillo L., Moreno N.F., Bordt A.S., Olson S.D, & Cox CS J.r. (2020). Human cord blood‐derived regulatory T‐cell therapy modulates the central and peripheral immune response after traumatic brain injury. Stem Cells Translational Medicine, 9(8), 903-916.

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Multiparameter Flow Cytometry of Immune Cells

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A total of 1 × 10⁶ mononuclear cells suspended in PBS were stained using incubation with 1 µg Fc Block (anti-CD16/32, Tonbo Biosciences) for 15 min at 4 °C. Cells were then stained with the following antibodies: CD45 (APC/Fire 750-Biolegend Clone: 13/2.3), CD11c (APC-Biolegend, Clone: N418) MHCII (PerCp Cy5.5-Biolegend, Clone: M5/114.15.2), CD88 (PE, Biolegend, Clone: 20/70), CD317 (BV421, Biolegend, Clone: 927), CD45.1 (FITC-Biolegend Clone: A20), and CD45.2 (BV605, Biolegend, Clone: 104) for 45 min at 4 °C. Events were recorded via BD FACS LSR Fortessa (The Moody Foundation Flow Cytometry Facility, UT Southwestern), equipped with Diva acquisition software (BD Bioscience). For FACS sorting, BD FACSARIA-II SORP (The Moody Foundation Flow Cytometry Facility, UT Southwestern) was utilized. Cells were gated according to morphology based on their side scatter (SSC) vs. forward scatter (FSC) distributions. Doublets were excluded (FSC-A vs. FSC-H and SSC-A vs. SSC-H, where A corresponds to area and H corresponds to height). Gating for FACS sorting consisted of singlet CD45⁺MHCII⁺CD88⁺CD317⁺ CD45.1⁺ cells or CD45⁺MHCII⁺CD88⁺CD317⁺CD45.2⁺ cells. In each sample, a minimum of 50 × 10³ live events were recorded. FlowJo software (BD Bioscience) was used for data analysis.

Manouchehri N., Salinas V.H., Hussain R.Z, & Stüve O. (2023). Distinctive transcriptomic and epigenomic signatures of bone marrow-derived myeloid cells and microglia in CNS autoimmunity. Proceedings of the National Academy of Sciences of the United States of America, 120(6), e2212696120.

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Multicolor Immune-Phenotyping of Microglia

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To specifically identify microglia and to characterize their pro- and anti-inflammatory phenotype, CD11b/c enriched cells were surface stained. The list of antibodies is summarized in our published optimized multicolor immune-phenotyping protocol^{26 (link)}. Each sample of the CD11b/c enriched cells was five times diluted with staining buffer (Biolegend), washed and divided to M1 (pro-inflammatory path) and M2 (anti-inflammatory path) and added with the appropriate antibody mix. Cells were incubated for 30 min at RT in the dark. After another wash, cells were added with the secondary antibody mix and Ghost live/dead reagent and incubated for 20 min at RT in the dark. Cells were finally washed, added to counting control beads (Cyto-Cal™) and run. Data was acquired with LSRII cytometer with Diva acquisition software (BD Biociences, San Jose, CA). Analysis was conducted using Kaluza vr. 1.5a software (Backman Coulter, Brea, CA). Fluorescence spillover compensation values were generated using VersaComp Antibody Capture Beads (Beckman Coulter, Inc.). tSNE analysis was performed for each panel separately using FlowJo vr. 10, with down sampling and tSNE plugin that included all markers (but not FS and SS).

Toledano Furman N., Gottlieb A., Prabhakara K.S., Bedi S., Caplan H.W., Ruppert K.A., Srivastava A.K., Olson S.D, & Cox CS J.r. (2020). High-resolution and differential analysis of rat microglial markers in traumatic brain injury: conventional flow cytometric and bioinformatics analysis. Scientific Reports, 10, 11991.

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Rat Microglia Immunophenotyping by Flow Cytometry

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In addition, at 72 hours, the rat microglia cells were collected and analyzed using flow cytometry. After cell culture supernatant was removed, the cells were washed with PBS and then detached from the culture plate with TrypLE Express (Thermo Fisher). The cells were washed, centrifuged at 400g, and resuspended in cell staining buffer. The isolated brain cells were immunophenotyped using the multiparametric flow cytometry panel (Figure 2) as we have previously described.¹⁰ The cells were then stained with the antibody mixture in Table 1 and incubated in the dark for 20 minutes, at which time 7-AAD was added. After 10 minutes, data were acquired with LSR-2 flow cytometer with Diva acquisition software (BD Biosciences, San Jose, CA). VersaComp Antibody Capture Beads (Beckman Coulter, Inc.) were used to create fluorescence spillover compensation values.

Caplan H.W., Prabhakara K.S., Toledano-Furman N.E., Zorofchian S., Kumar A., Martin C., Xue H., Olson S.D, & Cox CS J.r. (2020). Combination therapy with Treg and MSC enhances potency and attenuation of inflammation after traumatic brain injury compared to monotherapy. Stem cells (Dayton, Ohio), 39(3), 358-370.

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Immunophenotyping of Brain Cells

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At 14 days after injury, animals were euthanized, and brains were harvested. The ipsilateral hemispheres were processed as previously described above using the Neural Tissue Dissociation Kit and GentleMACS dissociator (Miltenyi Biotec).²⁸The isolated brain cells were immunophenotyped using the multiparametric flow cytometry panel (Figure 2) as we have previously described.¹⁰ The cells were then stained with the antibody mixture in Table 1 and incubated in the dark for 20 minutes, at which time 7-AAD was added. After staining, the red blood cells were lysed in a TQ-Prep (Beckman Coulter, Inc.). Finally, 25μL of counting control beads (Cyto-Cal^™) was added to each sample while vortexing. Data were acquired with LSR-2 flow cytometer with Diva acquisition software (BD Biosciences, San Jose, CA). VersaComp Antibody Capture Beads (Beckman Coulter, Inc.) were used to create fluorescence spillover compensation values.

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Phagocyte Depletion and Parasite Selection

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B6.RAG-1-/- mice were treated with 200uL i.v. of either saline (Sigma; negative control) or clodronate liposomes (clodronateliposomes.org). Flow cytometric analysis was carried out to assess the depletion of phagocytic cells. Single-cell suspensions of splenocytes were prepared, , and cells enumerated 24h after in vivo treatment. After staining with Zombie Aqua (Biolegend) for live/dead discrimination, cells were stained with monoclonal antibodies using appropriate combinations of fluorochromes (CD11c-BV786, CD169-BV605, F4/80-BV421, Ly6G-PerCPCy5.5, MHCII-FITC, Ter119-PE-Cy7, CD68-PE, Ly6c-APC-Cy7, CD11b-AF647, all from Biolegend). The samples were acquired on a BD Fortessa/X20 (BD Biosciences) using Diva acquisition software (BD Biosciences). FlowJo (Tree Star) software was used to analyze the data.
To determine the role of T-, B- and phagocytic cells in the selection of virulent parasites, B6.RAG-1-/- mice were treated with saline or clodronate liposomes as described above at days -1 and 4 after i.p. infection with 10⁵ iRBCs containing a mix of Neon-Green-expressing SBP parasites and mCherry-expressing MT parasites (see “Mixed infections” section).

Brugat T., Reid A.J., Lin J., Cunningham D., Tumwine I., Kushinga G., McLaughlin S., Spence P., Böhme U., Sanders M., Conteh S., Bushell E., Metcalf T., Billker O., Duffy P.E., Newbold C., Berriman M, & Langhorne J. (2017). Antibody-independent mechanisms regulate the establishment of chronic Plasmodium infection. Nature microbiology, 2, 16276.

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Comprehensive Immune Cell Phenotyping

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A total of 1 x 10 6 mononuclear cells suspended in PBS were stained using fixable viability dye (Zombie NIR-Biolegend), and incubated for 15 minutes at room temperature following manufacturer's recommendation. Cells were then washed using 2% FBS-PBS (FACs buffer) at 1500 rpm for 5 minutes, followed by incubation with 1 µg Fc Block (anti-CD16/32, Tonbo Biosciences) for 15 minutes at 4ºC. Cells were then stained with the following antibodies: CD45 (Alexa Flour 700-Biolegend, Clone: 30-F11), CD11b (V450-BD Horizon, Clone: M1/70), CD19 (BV510-Biolegend, Clone: MHCII (BV785, Biolegend, Clone: M5/114.15.2), CD49d (α4-integrin; FITC, Biolegend, Clone: R1-2), CD88 (PE, Bilegend, Clone: 20/70), CD317 (BV421, Biolegend, Clone: 927) CD26 (FITC, Biolegend, Clone: H194-112) for 45 minutes at 4ºC. Events were recorded via BD FACS LSR Fortessa (The Moody Foundation Flow Cytometry Facility, UT Southwestern), equipped with Diva acquisition software (BD Bioscience). Cells were gated according to morphology side scatter (SSC-A) vs forward scatter (FSC-A). Doublets were excluded (FSC-A vs FSC-H and SSC-A vs SSC-H). Live cells were selected using the viability dye. The complete gating strategy is presented in Supplementary Figure 1. In each sample a minimum of 50 x 10 3 live events were recorded. FlowJo software (BD Bioscience) was used for data analysis.

Manouchehri N., Hussain R.Z., Cravens P.D., Edelson B.T., Wu G.F., Cross A.H., Doelger R., Loof N., Eagar T.N., Forsthuber T.G., Calvier L., Herz J., & Stüve O. (2020). CD11c⁺CD88⁺CD317⁺myeloid cells are critical mediators of persistent CNS autoimmunity.

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