Data analysis software

Manufactured by FlowJo

Sourced in United States

FlowJo is a data analysis software designed for flow cytometry. It provides a user-friendly interface to visualize and analyze data generated from flow cytometry experiments. The core function of FlowJo is to process, display, and analyze flow cytometry data.

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29 protocols using data analysis software

Apoptosis Detection by Flow Cytometry

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Cells were collected and washed with PBS. Cells were diluted to a density of 1 × 10⁶ cells/ml and then stained with 100 μg/ml PI and fluorescein isothiocyanate–conjugated-annexin V in 500 μl of annexin V binding buffer for 15 min at room temperature. Samples were analyzed at 530 and 575 nm on a BD FACSCanto II (BD Biosciences, San Jose, CA). Data analysis was performed using FlowJo Data Analysis Software (FlowJo, Ashland, OR).

Moodley S., Hui Bai X., Kapus A., Yang B, & Liu M. (2015). XB130/Tks5 scaffold protein interaction regulates Src-mediated cell proliferation and survival. Molecular Biology of the Cell, 26(24), 4492-4502.

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Quantifying Sonoporated Cell Viability

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Flow cytometry was used to quantify the number of sonoporated cells for each treatment group, i.e., those cells displaying both PI uptake (permeabilization) and calcein-AM cleavage (viability). An LSRFortessa cytometer equipped with 561- and 488-nm excitation lasers (Becton Dickinson, Franklin Lakes, NJ, USA) was used for acquisition, and 30,000 events were recorded for each sonoporation treatment. For further details regarding acquisition settings, please see Additional file 1: Table S2.
The gating strategy employed to isolate sonoporated cells is described in full in Additional file 1: Information section and displayed in Additional file 2: Figure S1. Briefly, singlet cells were isolated from debris and doublet cells through initial gating steps. The viability of the chosen cell population was then confirmed by calcein fluorescence. Curly quadrant gates were applied to the calcein vs. PI fluorescence dot plots, with thresholds determined such that unstained control cells would be classified as both calcein and PI negative. The percent of cells in quadrant two (calcein and PI positive) was taken to be the sonoporation efficiency (i.e., percent of viable cells that were sonoporated). All data analysis was performed using FlowJo Data Analysis Software (FlowJo, LLC., Ashland, OR, USA).

Fix S.M., Novell A., Yun Y., Dayton P.A, & Arena C.B. (2017). An evaluation of the sonoporation potential of low-boiling point phase-change ultrasound contrast agents in vitro. Journal of Therapeutic Ultrasound, 5, 7.

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Detecting CAR Expression on T-Cells

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CAR transgene expression on T-cells was detected separately for each molecule using flow cytometry. Cell surface expression of FRP5 (HER2 CAR) and IL13 mutein were detected using conjugated Her2.Fc chimeric protein or IL13Rα2.Fc chimeric protein respectively (R&D Systems, Minneapolis, MN) followed by a PE-conjugated goat anti-human Fc (Thermo Fisher scientific, Waltham, MA). EPHA2 was detected using EPHA2 tagged with GST (Thermo Fisher Scientific, Waltham, MA) followed by anti-GST-PE (Abcam, Cambridge, MA). Additionally, FRP5 expression was also detected using AF647 anti-Mouse IgG, F(ab’)2 Fragment specific antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) as required. Flow capture was performed on an Accuri C6 (Becton Dickinson, Franklin Lakes, NJ). FlowJo data analysis software (FLOWJO, LLC, Ashland, OR) was used for analyses.

Donovan L.K., Delaidelli A., Joseph S.K., Bielamowicz K., Fousek K., Holgado B.L., Manno A., Srikanthan D., Gad A.Z., Van Ommeren R., Przelicki D., Richman C., Ramaswamy V., Daniels C., Pallota J.G., Douglas T., Joynt A.C., Haapasalo J., Nor C., Vladoiu M.C., Kuzan-Fischer C.M., Garzia L., Mack S.C., Varadharajan S., Baker M.L., Hendrikse L., Ly M., Kharas K., Balin P., Wu X., Qin L., Huang N., Stucklin A.G., Morrissy A.S., Cavalli F.M., Luu B., Suarez R., De Antonellis P., Michealraj A., Rastan A., Hegde M., Komosa M., Sirbu O., Kumar S.A., Abdullaev Z., Faria C.C., Yip S., Hukin J., Tabori U., Hawkins C., Aldape K., Daugaard M., Maris J.M., Sorensen P.H., Ahmed N, & Taylor M.D. (2020). Locoregional delivery of CAR T-cells to the cerebrospinal fluid for treatment of metastatic medulloblastoma and ependymoma. Nature medicine, 26(5), 720-731.

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Quantifying Th17 and Treg Cells

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Approximately 1 × 10⁶ cells were fixed in 2 mL 1× Human FoxP3 Buffer A (BD Biosciences) for 10 to 20 min at room temperature (RT) and protected from light. Fixed cells were washed with 2 mL Stain Buffer followed by centrifugation at 500g for 5 min. Cells were permeabilized in 0.5 mL 1× Human FoxP3 Buffer C (BD Biosciences) for 30 min at RT and protected from light. Cells were washed with Stain Buffer and resuspended to 1 × 10⁷ cells/mL in 100 μL aliquots. Antihuman antibodies (PerCP-Cy5.5-CD4, phycoerythrin (PE)-IL-17A, and Alexa Fluor647-FoxP3) or isotype controls (PerCP-Cy5.5-, PE-, and PEAlexa Fluor 647-conjugated mouse IgG1 κ isotype controls) were added to the cells (20 μL/test) and incubated for 40 min at RT protected from light. After staining, cells were washed twice with Stain Buffer and analyzed immediately using BD FACSCalibur flow cytometer (BD Biosciences). Approximately 20,000 to 30,000 cells were acquired and results were analyzed by FlowJo Data Analysis software (FlowJo LLC, Ashland, OR).

Zhu X., Li S., Zhang Q., Zhu D., Xu Y., Zhang P., Han J., Duan Z., Gao J, & Ou Y. (2018). Correlation of increased Th17/Treg cell ratio with endoplasmic reticulum stress in chronic kidney disease. Medicine, 97(20), e10748.

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Quantifying Nef-Mediated MHC-I Downregulation

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CEM-SS cells were transfected with 15 μg of plasmids expressing wild-type or mutated Nef-GFP plasmids. After 24 h, the cells were first stained for 1h at 4°C with an anti-MHCI antibody and then stained with a secondary PE-conjugated antibody in FACS staining buffer. Specific staining was analysed by two-colour flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson). FlowJo data analysis software (FLOWJO. LLC. http://www.flowjo.com/) was used to quantify the mean fluorescence intensity (MFI) of nontransfected and transfected cells. The MFI of the GFP-positive cells was used to calculate the % of activity of each mutant in reducing the surface expression of MCHI relative to wild-type Nef and expressed as the mean ± SD of three different assays done in duplicate.

Martínez-Bonet M., Palladino C., Briz V., Rudolph J.M., Fackler O.T., Relloso M., Muñoz-Fernandez M.A, & Madrid R. (2015). A Conserved GPG-Motif in the HIV-1 Nef Core Is Required for Principal Nef-Activities. PLoS ONE, 10(12), e0145239.

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Multiparametric flow cytometry analysis

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Right kidneys, lungs, and spleens were harvested and manually disrupted to create single-cell suspensions by pipetting through a 70 μM nylon filter (kidneys and lungs) or a 40 μM nylon filter (spleen). Cells were stained with combinations of surface and intracellular antibodies according to the manufacturers’ protocols. Antibodies were used to detect the following surface proteins: CD45−APC/Cy7, CD8−PerCP, CD4−V450, CD25−PE. Staining for intracellular FoxP3−APC and Ki67-FITC were done according to the manufacturers’ protocols. Dead cells were excluded using Fixable Viability Dye eFluor 506 stain (eBioscience [San Diego, CA]). Fc block (anti-mouse CD16/32 antibody, BioLegend, San Diego, CA) was used on all specimens. Antibodies were obtained from eBioscience or BD Biosciences (San Jose, CA). anti-PD-L1 (MIH5; eBioscience) was used to stain in vitro cultured Renca cells after co-culture with IFN-γ. Cells were analyzed using a BD LSR multi-channel flow cytometer (BD Biosciences) and FlowJo data analysis software (Ashland, OR).

O’Shaughnessy M.J., Murray K.S., La Rosa S.P., Budhu S., Merghoub T., Somma A., Monette S., Kim K., Corradi R.B., Scherz A, & Coleman J.A. (2017). Systemic antitumor immunity by PD-1/PD-L1 inhibition is potentiated by vascular-targeted photodynamic therapy of primary tumors. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(3), 592-599.

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Identifying Antigen-Specific T Cells

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All flow cytometry data were analyzed using the FlowJo data analysis software (version 10.8.1; FlowJo LLC). For antigen-specific T cell identification using combinatorial pMHC tetramer staining, we gated on single, live, CD3⁺, CD8⁺ lymphocytes and selected cells positive in two tetramer colors and negative in the remaining colors. For the IFN-γ detection, cells were gated as single, CD3⁺, CD8⁺ lymphocytes and double positive to CD8⁺ and IFN-γ.

Cavalluzzo B., Viuff M.C., Tvingsholm S.A., Ragone C., Manolio C., Mauriello A., Buonaguro F.M., Tornesello M.L., Izzo F., Morabito A., Hadrup S.R., Tagliamonte M, & Buonaguro L. (2024). Cross-reactive CD8+ T cell responses to tumor-associated antigens (TAAs) and homologous microbiota-derived antigens (MoAs). Journal of Experimental & Clinical Cancer Research : CR, 43, 87.

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Multiparametric Flow Cytometry of SVF Cells

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Purified SVF cells were blocked with anti-mouse CD16/32 antibody for 10 minutes on ice. For analysis of immune cell populations, cells were further incubated for 30 minutes on ice in the dark with the following anti-mouse antibodies obtained from BioLegend: APC-CD11b, APC-Cy7-CD11c, PE-CD206 or PE/Cy7-CD206, PerCP/Cy5.5-Ly6G, FITC-CD3, PerCP/Cy5.5-CD19, APC/Cy7-NK1.1. After antibody conjugation, cells were washed with Cell Staining Buffer and analyzed on a Becton-Dickinson LSRII analytical flow cytometer. For analysis of intracellular IL-6, purified SVF cells obtained by pooling the SVF of two AL mice were incubated with 1× Brefeldin A solution (BioLegend) for 4h in culture medium at 37°C. Blocking and cell surface staining were performed as described above followed by fixation and permeabilization using Fixation/Permeabilization Reagents and the protocol recommended by the manufacturer (eBioscience, San Diego, CA). Intracellular staining of IL-6 was performed using anti-mouse PE-IL-6 and negative control (PE-Rat IgG1, κ Isotype control). Cells were analyzed using iCyt Synergy Cell Sorter (Sony Biotechnology San Jose, CA). Analysis of flow cytometry data was performed using FlowJo data analysis software (FlowJo, LLC, Ashland, OR).

Starr M.E., MSteele A., Cohen D.A, & Saito H. (2016). Short-Term Dietary Restriction Rescues Mice From Lethalabdominal Sepsis and Endotoxemia, and Reduces the Inflammatory/Coagulant Potential of Adipose Tissue. Critical care medicine, 44(7), e509-e519.

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Phenotype Analysis of Mature Dendritic Cells

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Immature BMDCs were converted into mature cells by incubation with LPS for 16 hr. Mature DCs were harvested, and then reacted with rTvα-actinin 2 for additional 16 hr. The cells were then stained for 20 min on ice using the Live/dead Fixable Dead Cell Stain Kit (Invitrogen, Carlsbad, California, USA) to monitor their viability. Following 3 washes with PBS, the cells were reacted with one of these chemicals [allophycocyanin (APC)-conjugated anti-CD80, and phycoerythrin (PE)-conjugated anti-CD86, and peridinin chlorophyll (PerCP)-Cy 5.5-conjugated anti-mouse I-A/I-E for MHC class II] along with APC-eFluor780-conjugated anti-CD11c for 20 min. After 3 PBS-washes, the cells were prepared in 500 μl of PBS containing 1% BSA, and 0.1% sodium azide, and their fluorescence was measured by flow cytometry. The data analyses were performed using FlowJo data analysis software (FlowJo LLC, Ashland, Oregon, USA).

Lee H.Y., Kim J., Ryu J.S, & Park S.J. (2017). Trichomonas vaginalis α-Actinin 2 Modulates Host Immune Responses by Inducing Tolerogenic Dendritic Cells via IL-10 Production from Regulatory T Cells. The Korean Journal of Parasitology, 55(4), 375-384.

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Intracellular Cytokine Staining of Gag-Specific T Cells

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Spleens were harvested in Hank’s medium (Corning Cellgro) and cells were strained through a 70 μm filter for resuspension in complete RPMI [cRPMI: 10% FBS, penicillin (100 units/mL), streptomycin (100 μg/mL), 2 mM L-glutamine]. Red blood cells were lysed with ACK Lysing Buffer (Gibco) and splenocytes were resuspended in cRPMI at a concentration of 2×10⁷ cells/mL. Cells were plated at 1×10⁶ cells/well and incubated for 5 h in the presence of Golgi Plug (BD Biosciences) and 1 μg of one of five different immunodominant ZM96 Gag CD8 epitopes: LRSLYNTV (LRS8), VIPMFTAL (VIP8), AMQMLKDT (AMQ8), YSPVSILDI (YSP9), EVKNWMTDTL (EVK10) (synthesized by GenScript and resuspended according to manufacturer’s protocol). Cells were stained with fluorescently conjugated antibodies: CD8-Pacific Blue, TNFα-FITC, and IFNγ-PE (BD Biosciences). Fixation and permeabilization was performed with a Cytofix/Cytoperm Kit (BD Biosciences) with final resuspension in FACS Buffer (1% FBS in 1× PBS). Samples were analyzed by flow cytometry on an LSR Fortessa. Data was analyzed using FlowJo Data Analysis Software (FlowJo, LLC; Ashland, OR).

Meador L.R., Kessans S.A., Kilbourne J., Kibler K.V., Pantaleo G., Roderiguez M.E., Blattman J.N., Jacobs B.L, & Mor T.S. (2017). A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles. Virology, 507, 242-256.

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