Fortessa x20

Manufactured by BD

Sourced in United States, Germany, France, Canada

The Fortessa X20 is a flow cytometry instrument designed for advanced cell analysis. It is capable of detecting and measuring multiple parameters of individual cells within a sample. The core function of the Fortessa X20 is to provide high-performance data acquisition and analysis for researchers and clinicians working in the fields of immunology, oncology, and cell biology.

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Close spelling variants of this product

Fortessa X20 analyzer (4 citations) Fortessa LSR X20 (2 citations) Fortessa X20 flow cytometer (111 citations) Fortessa X20 cytometer (43 citations) BD FACS Fortessa X20 analyzer (2 citations) FACS LSR Fortessa X20 (20 citations) FACS Fortessa X20 (18 citations) Fortessa (1232 citations)

346 protocols using fortessa x20

Vaccine Formulations Enhance Antigen-Specific T Cells

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Mice (C57BL/6, n = 5 in each group from two pooled experiments) were injected
intravenously with different vaccine formulations, such as multi-LP/α-GalCer
+ TRP2-mRNA, multi-LP/TRP2-mRNA, multi-LP/TRP2-mRNA + free α-GalCer,
free α-GalCer + TRP2-mRNA, and PBS as a negative control. The
vaccine formulations were prepared using 10 μg of TRP2-mRNA
and 0.5 μg of α-GalCer. Blood was collected from vaccinated
mice by saphenous bleeding and erythrocytes were removed by addition
of ACK lysis buffer (Quality Biological). peripheral blood mononuclear
cells (PBMCs) were stimulated with 1 μg/mL of TRP2-peptide (SVYDFFVWL)
for 6 h in the presence of a protein transport inhibitor, Brefeldin
A (BD GolgiPlug). After incubation, cells were washed and stained
with Abs for surface markers-TCRβ, CD8, and V450/viability dye.
Cells were then washed, fixed, and permeabilized with a BD Fixation/Permeabilization
Solution Kit and stained with anti-IFN-γ. The cells were washed,
resuspended in FACS buffer (1× PBS and 0.5% BSA), and analyzed
by flow cytometry (BD Fortessa X-20).
SVYDFFVWL-specific tetramer
(MBL International) staining was carried out by incubation at room
temperature for 10 min in FACS buffer. After washing, PBMCs were stained
with anti-TCRβ, anti-CD8 clone KT15, and V450/viability for
30 min at 4 °C in FACS buffer. Then, the samples were analyzed
by flow cytometry (BD Fortessa X-20).

Guevara M.L., Jilesen Z., Stojdl D, & Persano S. (2019). Codelivery of mRNA with α-Galactosylceramide Using a New Lipopolyplex Formulation Induces a Strong Antitumor Response upon Intravenous Administration. ACS Omega, 4(8), 13015-13026.

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Identification of Human and Mouse Basophils

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For human studies, thawed PBMCs were stained with Zombie UV viability dye (1:500; Biolegend) at room temperature for 20 minutes, washed and then stained with primary antibodies on ice for 30 minutes before being acquired on a BD Fortessa X-20 (BD Biosciences). Human Basophils were defined as live CD123⁺ FcεRIα⁺ cells that lacked expression of c-Kit and lineage (Lin) markers CD3, CD4, CD19, CD14, CD34, and CD56. arker CD203c was included in the primary antibodies to reveal the physiological state of human basophils. For animal studies, 50–100 µL of blood was collected into EDTA coated tubes, followed by RBC lysis using RBC lysis buffer (Sigma-Aldrich) at room temperature for 5 minutes twice and washed by PBS once. All cells were stained with Zombie UV viability dye (1:500; Biolegend) for viability at room temperature for 20 minutes, followed by primary antibodies on ice for 30 minutes prior to data acquisition on a BD Fortessa X-20 (BD Biosciences). Basophils were defined as live CD49b⁺ FcεRIα/IgE⁺ cells that were negative for expression of c-Kit and Lin markers CD3e, CD5, CD11c, CD19, and NK1.1. The mouse basophil canonical activation marker CD200R was also stained. All flow cytometry data were analyzed with Flowjo v10 software (Tree Star).

Wang F., Trier A.M., Li F., Kim S., Chen Z., Chai J.N., Mack M.R., Morrison S.A., Hamilton J.D., Baek J., Yang T.L., Ver Heul A.M., Xu A.Z., Xie Z., Dong X., Kubo M., Hu H., Hsieh C.S., Dong X., Liu Q., Margolis D.J., Ardeleanu M., Miller M.J, & Kim B.S. (2021). A Basophil-Neuronal Axis Promotes Itch. Cell, 184(2), 422-440.e17.

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Evaluation of EuroFlow IMC Tube

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The technical performance of the EuroFlow IMC tube was evaluated in a Fortessa X20 (BD) for different anti-coagulants (EDTA vs. sodium heparin) (assessed in 7 paired PB samples stained with versions 2 (n=3) and 3 (n=4) of the IMC tube; Table 1), immediate vs. delayed (storage at RT for 6h, 12h and 24h) (n=3; version 3) and fresh vs. frozen (n=3; version 4) staining of (EDTA anti-coagulated) PB samples. To compare the performance of the EuroFlow IMC tube in different instruments (i.e., conventional vs. spectral flow cytometers), PB samples from 5 donors were stained with version 4 of the tube (Table 1) and measured in parallel in a 4-laser Fortessa X20 (BD) conventional flow cytometer (405nm, 488nm, 561nm and 640nm lasers) and a 3-laser Aurora (Cytek) (405nm, 488nm, 640nm) spectral flow cytometer.

van der Pan K., de Bruin-Versteeg S., Damasceno D., Hernández-Delgado A., van der Sluijs-Gelling A.J., van den Bossche W.B., de Laat I.F., Díez P., Naber B.A., Diks A.M., Berkowska M.A., de Mooij B., Groenland R.J., de Bie F.J., Khatri I., Kassem S., de Jager A.L., Louis A., Almeida J., van Gaans-van den Brink J.A., Barkoff A.M., He Q., Ferwerda G., Versteegen P., Berbers G.A., Orfao A., van Dongen J.J, & Teodosio C. (2022). Development of a standardized and validated flow cytometry approach for monitoring of innate myeloid immune cells in human blood. Frontiers in Immunology, 13, 935879.

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Multiparameter Flow Cytometry Analysis of Immune Cell Populations

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Organs were collected from WT, CD45.1, CD45.2, OT-I, or CD8β-deficient mice and single-cell suspensions prepared using standard protocols^. Following removal of red blood cells using ACK, nonspecific receptors were blocked with monoclonal antibody (mAb) 2.4G2, before cells (1–5 × 10⁶) were stained with mAb to mouse NKp46 (29A1.4; BioLegend), CD3 (17A2; BD Biosciences), TCRγδ (GL3, BioLegend), TCRαβ (H57-597, BioLegend), CD8α (53-6.7; BD Biosciences), CD8β (H35-17.2; BD Biosciences). Alternatively cells were stained with the lectins peanut agglutinin (Vector Laboratories), sambucus nigra lectin (Vector Laboratories), or maackia amurensis lectin II (Vector Laboratories) before detecting using Streptavidin (BD Biosciences). Cells stained with tetramers were fixed in 2% paraformaldehyde for 15 min and washed twice with FACS buffer (1% FCS/PBS) before being resuspended in FACS buffer. All other FACS combinations were acquired unfixed. For acquisition, events were electronically gated on FSC-A versus FSC-H (singlets), followed by FSC-A and SSC-A (to exclude doublets and debris). Among the remaining population, at least 5000 electronic events of interest were collected using an LSR-II or X- 20 Fortessa (BD Biosciences).

Goodall K.J., Nguyen A., Andrews D.M, & Sullivan L.C. (2021). Ribosylation of the CD8αβ heterodimer permits binding of the nonclassical major histocompatibility molecule, H2-Q10. The Journal of Biological Chemistry, 297(4), 101141.

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Dual Fluorescence Reporter Transfection

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Cells were transfected with equal concentration of the dual GFP–ChFP fluorescence reporter containing K20 internal repeat [poly(A)] or K0 control (Sundaramoorthy et al., 2017 (link)) in 293T cells. Fluorescence levels were measured using an X-20 Fortessa (BD) instrument, as described previously (Sundaramoorthy et al., 2017 (link)).

Sundaramoorthy E., Ryan A.P., Fulzele A., Leonard M., Daugherty M.D, & Bennett E.J. (2021). Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection. Journal of Cell Science, 134(8), jcs257188.

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Flow Cytometric Analysis of CHO Cells

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CHO ART2.2-CD8αα and CHO ART2.2-CD8αβ cells were routinely cultured and liberated from the tissue culture flask with the use of TrypLE Express (Thermo Fisher Scientific). The cells were stained with the antibodies CD8α or CD8β. Cells stained with tetramers were fixed in 2% paraformaldehyde and washed twice with FACS buffer (1% FCS/PBS) before being resuspended in FACS buffer. All other FACS combinations were acquired unfixed. For acquisition, events were electronically gated on FSC-A versus FSC-H (singlets), followed by FSC-A and SSC-A (to exclude doublets and debris). Among the remaining population at least 10,000 electronic events of interest were collected using an LSR-II or X- 20 Fortessa (BD Biosciences).

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Flow Cytometry Data Acquisition and Analysis

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Data were acquired on a LSRII or X-20 Fortessa (BD Biosciences). Data were exported as FCS files and analyzed using FlowJo software (TreeStar, version 10). Compensation for multicolor experiments was set using single mAb-stained beads, and cytometer setup and tracking beads were run daily.

Sim M.J., Rajagopalan S., Altmann D.M., Boyton R.J., Sun P.D, & Long E.O. (2019). Human NK cell receptor KIR2DS4 detects a conserved bacterial epitope presented by HLA-C. Proceedings of the National Academy of Sciences of the United States of America, 116(26), 12964-12973.

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Plasma Cytokine Profiling in ABA Mice

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Blood was collected from tail tip at 6 weeks of age, 5 days prior to the commencement of behavioral testing. Approximately, 350 μl of whole blood was collected into EDTA‐coated tubes (Microvette; Brand), which were centrifuged for 10‐min (8000 rpm, 4°C) within 30 min of collection and plasma separated and stored at −80°C until use. Following exposure to ABA conditions, and including 7 days of ad libitum food access and body weight recovery to >100% baseline to ensure that effects of susceptibility to ABA were not confounded by the acute effects of starvation, blood was collected via cardiac puncture. A custom rat multianalyte LEGENDplex bead‐based immunoassay kit was used to examine cytokine concentrations in plasma samples (LEGENDplex; BioLegend) that targeted six cytokines concurrently (IL‐6, IL‐10, IL‐4, IL‐1β, TNF‐α, RANTES). These analytes were selected based on their previously reported elevation in human AN patients and/or ABA mice. Plasma samples were screened with the LEGENDplex assay kit as per manufacturer's instructions, and the readout measurement acquired using a Fortessa X‐20 flow cytometer (Becton Dickinson [BD]). Data were analyzed using LEGENDplex Data Analysis Software (v8.0; BioLegend).

Milton L.K., Patton T., O'Keeffe M., Oldfield B.J, & Foldi C.J. (2022). In pursuit of biomarkers for predicting susceptibility to activity‐based anorexia in adolescent female rats. The International Journal of Eating Disorders, 55(5), 664-677.

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CFSE-based Proliferation Assay for T-cell Activation

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Peripheral blood mononuclear cell (PBMC) were labeled with carboxyfluorescein succinimidyl ester (CFSE, 200 nM; interchim, Montluçon, France) and seeded in 96-well round-bottom plates at 2.10⁵/well. Cells were cultured in RPMI 1640 supplemented with 10% human AB serum (Biowest, Nuaillé, France) and anti-CD3 and anti-CD28 monoclonal antibodies (0.6 µg/mL, Sanquin, Amsterdam, The Netherlands). After 4 days of culture, cells were harvested and labeled with CD2 PC7, CD8 APC (Beckman Coulter, Miami, FL), CD4 BUV496, CD14 BV605 (Becton Dickinson, San Jose, CA, USA) and DAPI (Sigma-Aldrich, St Louis, MO, USA). CFSE dilution was assessed on DAPI^neg viable T-cells by flow cytometry on Fortessa X-20 (Becton Dickinson) and the results were analyzed with ModFit LT software.

Frerou A., Lesouhaitier M., Gregoire M., Uhel F., Gacouin A., Reizine F., Moreau C., Loirat A., Maamar A., Nesseler N., Anselmi A., Flecher E., Verhoye J.P., Le Tulzo Y., Cogné M., Roussel M., Tarte K, & Tadié J.M. (2021). Venoarterial extracorporeal membrane oxygenation induces early immune alterations. Critical Care, 25, 9.

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Flow Cytometry of Reporter Strains

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Cells containing HSS reporters were grown for flow cytometry experiments as described (Greenstein et al., 2020 (link)). Flow cytometry was performed using a Fortessa X20 dual machine (Becton Dickinson) and high-throughput sampler (HTS) module. Depending on strain growth and sample volume, data from approximately 4,000–100,000 cells were collected. Fluorescence detection, compensation, and data analysis were done as described (Al-Sady et al., 2016 (link); Greenstein et al., 2018 (link)). 2D-density histogram plots were generated as described previously (Greenstein et al., 2018 (link)) with the following exceptions: Data from biological replicates were merged together prior to plotting. Hexbin plots were generated in R via the ggplot2 package. The guide-lines for cutoff values of “off” and “on” states for Green and Orange were determined using mean of a Red-Only control strain plus 3 times the standard deviation (SD) and mean of clr4Δ (after removal of color-negative cells) minus 1 SD value respectively. For the spt16–1 experiment, the fraction of cells below the “off” threshold for both “green” and “orange” were calculated for each biological replicate independently.

Murawska M., Greenstein R.A., Schauer T., Olsen K.C., Ng H., Ladurner A.G., Al-Sady B, & Braun S. (2021). The histone chaperone FACT facilitates heterochromatin spreading by regulating histone turnover and H3K9 methylation states. Cell reports, 37(5), 109944.

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