Lsr fortessa cytometer

Manufactured by BD

Sourced in United States, Poland, Germany, France, Australia, United Kingdom

The BD LSR Fortessa cytometer is a flow cytometry instrument designed for multi-parameter analysis of single cells. It is capable of detecting and analyzing a wide range of fluorescent signals from cells or particles. The core function of the LSR Fortessa is to provide researchers with high-performance flow cytometry capabilities for applications such as immunophenotyping, cell sorting, and fluorescence-activated cell analysis.

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Lab products found in correlation

Facsdiva software, by BD (44 mentions) Cytofix cytoperm kit, by BD (14 mentions) Golgiplug, by BD (12 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (12 mentions) Perm wash buffer, by BD (9 mentions) Facsdiva, by BD (9 mentions) Golgistop, by BD (9 mentions) 24 well plate, by Sarstedt (9 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (8 mentions) Propidium iodide, by Merck Group (8 mentions) Fitc annexin 5 apoptosis detection kit 1, by BD (7 mentions) Cytofix cytoperm buffer, by BD (6 mentions) Ionomycin, by Merck Group (6 mentions) Facscanto 2, by BD (6 mentions) Triton x 100, by Merck Group (6 mentions) Facsaria, by BD (6 mentions) Fcr blocking reagent, by Miltenyi Biotec (5 mentions) Phorbol myristate acetate (pma), by Merck Group (5 mentions) Fixable viability dye efluor 780, by Thermo Fisher Scientific (5 mentions) Live dead aqua, by Thermo Fisher Scientific (5 mentions)

485 protocols using lsr fortessa cytometer

Ki67-Hoechst Assay for Cell Cycle Analysis

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For Ki67-Hoechst assays, CD34+ cells with or without editing were first stained with fixable viability stain 660 (1:1000, BD) for 5 min in 37°C and were fixed with Cytofix/Cytoperm buffer (BD) for 15 min in 4°C. Cells were stained with FITC-anti-KI67 (1:25, BD, 556027) for 2hours-overnight in Permwash buffer (BD), then with Hoechst 33342 (1:5000; Life Technologies) for 5 min at RT. Samples were sorted on a Aria Fusion Cell Sorter (BD) or analyzed on a LSR Fortessa cytometer (BD). For assessment of immunophenotypic markers together with cell cycle analysis, human CD34+ cells with or without editing were stained with Percp-Cy5.5-anti-CD34 (1:50) and PE-Cy7-anti-CD38 (1:50) for 30 min in 4°C before they were stained with fixable viability stain and fixed. For assessment of cell cycle status without fixation (live cell cycle status), cells with or without editing were stained with Hoechst 33342 (1:1000, Invitrogen) for 45min in 37°C, and then were stained with Pyronin Y (1:20,000, Invitrogen) for additional 15 min in 37°C or were just stained with Pyronin Y for 15 min in 37°C. Samples were then sorted on Aria Fusion Cell Sorter (BD) or analyzed on LSR Fortessa cytometer (BD).

Shin J.J., Schröder M.S., Caiado F., Wyman S.K., Bray N.L., Bordi M., Dewitt M.A., Vu J.T., Kim W.T., Hockemeyer D., Manz M.G, & Corn J.E. (2020). Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells. Cell Reports, 32(9), 108093.

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Pluripotent Stem Cell Cycle and Hematopoietic Progenitor Analysis

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For cell-cycle analysis of pluripotent stem cells expressing the marker Tra-1-60 or Tra-1-81, cultures of iPSCs growing on MEFs were collected using TrypLE Express (Invitrogen). Cells were incubated with a Tra-1-60 or Tra-1-81 antibody in 1% PBS-BSA for 30 minutes followed by incubation with a secondary Alexa-Fluor 488 anti-mouse antibody (Invitrogen) for another 30 minutes. After this incubation, cells were fixed in aldehyde-based fixative overnight. A Click-iT EdU flow cytometry analysis kit (Invitrogen) was used to analyze the proliferation of the Tra-1-60 or Tra-1-81 positive population of cells following manufacturer’s recommendations. For cell-cycle analysis of HPCs, differentiated cells were stained with anti-human CD34-PE and anti-human CD43-FITC, fixed and permeabilized using the BD Cytofix/Cytoperm™ kit, stained with Alexa Fluor® 647 anti-human Ki-67 and DAPI, and then analyzed on a BD LSRFortessa cytometer. For apoptosis analysis of HPCs, differentiated cells were stained with anti-human CD34-PE, anti-human CD43-APC and Annexin V FITC, and analyzed on a BD LSRFortessa cytometer.

Liu G.H., Suzuki K., Li M., Qu J., Montserrat N., Tarantino C., Gu Y., Yi F., Xu X., Zhang W., Ruiz S., Plongthongkum N., Zhang K., Masuda S., Nivet E., Tsunekawa Y., Soligalla R.D., Goebl A., Aizawa E., Kim N.Y., Kim J., Dubova I., Li Y., Ren R., Benner C., del Sol A., Bueren J., Trujillo J.P., Surralles J., Cappelli E., Dufour C., Esteban C.R, & Belmonte J.C. (2014). Modeling Fanconi Anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs. Nature communications, 5, 4330.

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Mouse Stomach Cell Immunophenotyping

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The mouse stomach cells were blocked with 2 μL/10⁶ cells of Fc Block (BD) in 100 μL fluorescence-activated cell sorter (FACS) buffer (0.5% FBS, 1 mmol/L EDTA, 0.05% NaN₃ in PBS) for 10 minutes at 4°C, followed by washing with FACS buffer to remove Fc Block residue. Cell surface staining was performed in FACS buffer containing antibody cocktails (PerCP [Peridinin chlorophyll] /Cy [Cyanine] 5.5-CD45, APC [Allophycocyanin]/Cy7-CD11b, and APC-F4/80; PerCP/Cy5.5-CD45, APC/Cy7-CD11b, APC-F4/80, Pacific Blue–MHCII, and PE/Cy7-CD93; PerCP/Cy5.5-F4/80, APC/Cy7-CD11b, Pacific Blue–MHCII, PE/Cy7-CD93, APC-CD64, APC-CD21/35, APC-CD204, APC-CD14, and APC-VSIG4) on ice for 1 hour. After washing with FACS buffer 3 times, the cells were subjected to flow cytometry with a BD LSR Fortessa cytometer and data were analyzed with FlowJo software.
For intracellular staining, the Fc-blocked cell surface was stained with antibody cocktails on ice for 1 hour. After washing with FACS buffer, the cells were fixed using Cytofix/Cytoperm solution (BD) for 20 minutes on ice, followed by washing with Perm/Wash solution (BD). Intracellular staining was performed using PE-Il1β (BioLegend) for 1 hour on ice. The cells were washed again with Perm/Wash solution twice and analyzed by flow cytometry using a BD LSR Fortessa cytometer.

Kim K.H., Park J., Cho Y., Cho S.Y., Lee B., Jeong H., Lee Y., Yi J.W., Oh Y., Lee J.J., Wang T.C., Lim K.M, & Nam K.T. (2022). Histamine Signaling Is Essential for Tissue Macrophage Differentiation and Suppression of Bacterial Overgrowth in the Stomach. Cellular and Molecular Gastroenterology and Hepatology, 15(1), 213-236.

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Flow Cytometric Analysis of NK Cell Phenotypes

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Cell surface staining was performed on NK cells after 18h of incubation with Control-CM, ALG-CM, or 3mM CaCl₂ in R10 complete medium. Cells were stained in staining buffer with fluorochrome-linked monoclonal antibodies: anti-CD14-FITC (BD Biosciences), anti-CD3-FITC (Biolegend), anti-CD56-AF700 (BD Biosciences), anti-CD16-BUV395 (BD Biosciences), anti-CD69-BUV737 (BD Biosciences), anti-DNAM-1-PE-Vio770 (Miltenyi), anti-NKp30- anti-BV421 (BD Biosciences), anti-NKp46-BV786 (BD Biosciences), anti-NKG2D-BV650 (BD Biosciences), anti-KIR2D-PE (Miltenyi), anti-KLRG1-APC-Vio770 (Miltenyi), anti-CD95-APC (Biolegend), anti-CD85j-PE-Cy5 (BD Biosciences), anti-CD158e1/e2 PerCP-Vio700 (Miltenyi), anti-TIGIT-BV605 (Biolegend), and Fixable Viability Dye eFluor 506. The expression of activating or inhibitory receptors was analyzed on NK cell subsets based on CD16 and/or CD56 expression. Samples were analyzed using an LSRFortessa cytometer (BD Biosciences), and results were generated by FlowJo software.

Adib Y., Boy M., Serror K., Dulphy N., des Courtils C., Duciel L., Boccara D., Mimoun M., Samardzic M., Bagot M., Bensussan A, & Michel L. (2023). Modulation of NK cell activation by exogenous calcium from alginate dressings in vitro. Frontiers in Immunology, 14, 1141047.

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Multiparametric Flow Cytometry Analysis

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Following red blood cell lysis, SVF, blood and the BM (or In vitro-generated, BM-Derived monocytes) were washed with FACS buffer (PBS, 1% (v/v) heat-inactivated FBS, 0.2% (w/v) BSA), stained with Aqua Live/Dead Antibody (ThermoFisher, L34957) for 15 min in the dark, washed with FACS buffer again, blocked with anti-mouse CD16/32 antibody (eBioscience, 14-0161-86) for 15 min in the dark following a 30 min incubation with corresponding antibody for each tissue (see Table 1 for list of antibodies). The samples were then fixed, read on LSR Fortessa cytometer (BD Biosciences) and data gated based on appropriate fluorescence minus one (FMO) controls, and analyzed on the FlowJo software version 10 (Becton, Dickinson & Company).

Boroumand P., Prescott D.C., Mukherjee T., Bilan P.J., Wong M., Shen J., Tattoli I., Zhou Y., Li A., Sivasubramaniyam T., Shi N., Zhu L.Y., Liu Z., Robbins C., Philpott D.J., Girardin S.E, & Klip A. (2022). Bone marrow adipocytes drive the development of tissue invasive Ly6Chigh monocytes during obesity. eLife, 11, e65553.

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Multiparameter Flow Cytometry Analysis

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LSR Fortessa cytometer (BD Biosciences) equipped with 6 lasers (ultraviolet, 355 nm; violet, 405 nm; blue, 488 nm; green, 552 nm; orange, 592 nm; and red laser, 628 nm) and BD FACSDiva software (v.8.0.1, BD Biosciences) were used for sample acquisition. Compensation was calculated using bead standards for each fluorochrome (anti-Mouse Ig, κ/Negative Control Compensation Particles Set, BD Biosciences) and barcoded cells with the highest concentration of each dye. An unstained sample was used as negative control for setting PMT voltages, and all samples were run using the same PMT voltages. A minimum of 300,000 lymphocytes were recorded. FlowJo software (v.10.0.7b, Treestar, Ashland, OR, USA) was employed for post-acquisition compensation and conventional flow cytometric analysis. Compensation matrix was exported from FlowJo as a CSV file (Supporting Information S1). FCM data are shown using biexponential transformation.

Tsai W.L., Vian L., Giudice V., Kieltyka J., Liu C., Fonseca V., Gazaniga N., Gao S., Kajigaya S., Young N.S., Biancotto A, & Gadina M. (2019). High Throughput pSTAT Signaling Profiling by Fluorescent Cell Barcoding and Computational Analysis. Journal of immunological methods, 477, 112667.

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Multiparameter Flow Cytometry Acquisition

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Sample acquisition was implemented on a LSR Fortessa cytometer (BD Biosciences) equipped with ultraviolet (UV, 355 nm), violet (407 nm), blue (488 nm), green (532 nm), and red (633 nm) lasers, and BD FACSDiva software (v.8.0.1, BD Biosciences). Compensation was performed using a bead standard for each fluorochrome (anti-Mouse Ig, κ/Negative Control Compensation Particles Set, BD Biosciences) and barcoded cells with the highest concentration of each dye. Samples stained with the same FCB dyes and/or antibody combination were run using the same PMT voltages. A minimum of 10,000–30,000 lymphocytes was recorded. Post-acquisition compensation and flow cytometric analysis were performed using FlowJo software (v.10.0.7b, Treestar, Ashland, OR, USA). Lymphocytes were identified based on forward scatter area (FSC-A) vs SSC-A and doublet exclusion (FSC-A vs FSC-H), and then single cells were gated for FCB dye channels (Supplemental Fig. 2A). Additional gating strategies for viability dye and antibody staining are shown in Supplemental Fig. 2B–D.

Giudice V., Feng X., Kajigaya S., Young N.S, & Biancotto A. (2017). Optimization and Standardization of Fluorescent Cell Barcoding for Multiplexed Flow Cytometric Phenotyping. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 91(7), 694-703.

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Immune Cell Isolation and Analysis

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Peritoneal lymphocytes were obtained by peritoneal lavage with PBS, or cells were isolated from spleen or bone marrow as previously indicated (28 (link), 29 (link)). Equal numbers of cells were incubated with live dead viability dye (Life technologies) and then stained with antibodies against surface antigens in PBS with 3% FBS. Following washing, cells were either analyzed directly or treated with cytofix/cytoperm buffer (BD biosciences) and then stained with antibodies against intracellular antigens. The antibodies used are listed in Supplemental Table 1. Samples were run on a FACSCalibur or LSR Fortessa cytometer (BD Biosciences) and analyzed with Flowjo software (Treestar). Sorting was performed on a MoFlo Astrios (Beckman-Coulter). AnnexinV assays were performed according to manufacturer instructions (BD Biosciences) except that annexinV antibody was used at a 1:100 dilution. BrdU incorporation assays were performed by incubating cells in medium containing 10uM BrdU and staining as instructed (BD Biosciences).

DeMicco A., Naradikian M.S., Sindhava V.J., Yoon J.H., Gorospe M., Wertheim G.B., Cancro M.P, & Bassing C.H. (2015). B cell-intrinsic expression of the HuR RNA-binding protein is required for the T cell-dependent immune response in vivo. Journal of immunology (Baltimore, Md. : 1950), 195(7), 3449-3462.

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Quantifying Lung Metastasis in Mammary Tumor Mice

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Metastasis was analyzed post mortem in the lung of mice bearing large mammary tumor grafts (around 700~800 mm³). In brief, lungs were collected, minced in a Petri dish on ice and sequentially digested in Hanks balanced salt solution (HBSS) containing collagenase (1.25 mg/ml) and hyaluronidase (125 μg/ml) for 30 min, trypsin-EDTA for 3 min, and dispase (5 mg/ml) and DNase (10 mg/ml) for 3 min. Neutralization with 1% FBS containing HBSS was conducted between each enzymatic digestion, following centrifugation at 5,000 rpm. The final cell suspension was filtered through a 40 μm cell strainer. Red blood cells were solubilized with red cell lysis buffer (Pharm Lyse, BD Biosciences) and the resulting suspension was filtered through a cell strainer to produce single-cell suspensions. Dissociated cells were washed twice in PBS and incubated with DAPI (blue). For analysis, a typical forward- and side-scatter gate was set to exclude dead cells and aggregates; a total of 2 x 10⁶ events in the gate were collected using the BD Biosciences LSR Fortessa cytometer. tdTomato positive cells were quantified using Flowjo software.

Xu Q., Zhang J., Telfer B.A., Zhang H., Ali N., Chen F., Risa B., Pearson A.J., Zhang W., Finegan K.G., Ucar A., Giurisato E, & Tournier C. (2021). The extracellular-regulated protein kinase 5 (ERK5) enhances metastatic burden in triple-negative breast cancer through focal adhesion protein kinase (FAK)-mediated regulation of cell adhesion. Oncogene, 40(23), 3929-3941.

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Cell Surface Marker Staining and Viability

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Cells were stained for surface markers for 30 mins at 4°C, protected from light. DAPI (Invitrogen, #D1306, working concentration: 1 μg/mL) was added prior to running to assess viability. Samples were analyzed using a LSR Fortessa cytometer (BD Biosciences) and FlowJo software.

Minns D., Smith K.J., Hardisty G., Rossi A.G, & Gwyer Findlay E. (2021). The Outcome of Neutrophil-T Cell Contact Differs Depending on Activation Status of Both Cell Types. Frontiers in Immunology, 12, 633486.

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