4 laser fortessa

Manufactured by BD

The 4-Laser Fortessa is a flow cytometer that utilizes four lasers to detect and analyze multiple parameters of cells or particles in a sample. It is designed to provide high-performance data acquisition and analysis capabilities for a wide range of applications in life science research.

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Lab products found in correlation

Facsdiva software, by BD (3 mentions) Cytofix cytoperm, by BD (3 mentions) Normal rat serum, by STEMCELL (3 mentions) Facsaria 3, by BD (2 mentions) Live dead stain, by Thermo Fisher Scientific (2 mentions) 5 laser lsrii, by BD (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Zombie aqua fixable viability dye, by BioLegend (2 mentions) Mt10040cv, by Merck Group (1 mentions) Foxp3 transcription factor staining kit, by Cytek Biosciences (1 mentions) Cd11b, by BD (1 mentions) Human fc block, by BD (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Ficoll paque, by GE Healthcare (1 mentions) Jurkat, by Leibniz Institute DSMZ (1 mentions) Lympholyte mammal cell separation media, by Cedarlane (1 mentions) Rpmi 1640, by Irvine Scientific (1 mentions) Anti human cd20 clone 2h7, by BioLegend (1 mentions)

Close spelling variants of this product

Fortessa (1232 citations) LSR Fortessa 4 laser (6 citations) 4-laser Fortessa™ flow cytometer (2 citations) 3-or 4-Laser Fortessa (2 citations) LSR Fortessa 4-laser flow cytometer (2 citations)

12 protocols using 4 laser fortessa

Dissociation and Flow Cytometry of Tumor and Lung Samples

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Tumors and lungs were minced and dissociated in RPMI-1640 media (Corning #MT10040CV) containing 2.5% FBS, 1 mg/ml collagenase IA (Sigma-Aldrich #C9891), and 0.25 mg/ml DNase I (Sigma-Aldrich #DN25) for 45 minutes at 37°C. Digested tissue was then filtered through a 70-µm strainer, and red blood cells were lysed using ACK Lysis Buffer (KD Medical #RGF-3015). Samples were washed with PBS and stained with Ghost Dye Violet V510 (Tonbo Biosciences #13-0870) to exclude dead cells. After washing with buffer (0.5% BSA, 2mM EDTA in PBS), samples were blocked in αCD16/32 mouse Fc block (Tonbo Biosciences #70-0161) and stained for extracellular proteins using an antibody master mix made in buffer. After washing with buffer, cells were fixed with 2% PFA. For FoxP3 intracellular staining, cells were permeabilized using the FoxP3 Transcription Factor Staining Kit (Tonbo Biosciences #TNB-0607-KIT) per manufacturer protocol. Flow cytometry data was obtained on a BD 4-laser Fortessa using BD FACS Diva software v8.0.1 and analyzed using FlowJo software v10.6.1. Fluorescence minus one (FMO) samples were used as gating controls when needed. Antibodies used in flow panels are detailed in
Table 1, and gating strategies used in analysis are detailed in
Table 2. Each data point is generated after analyzing at least 5×10
⁵ viable cells from a specimen from an individual mouse.

Shiuan E., Inala A., Wang S., Song W., Youngblood V., Chen J, & Brantley-Sieders D.M. (2020). Host deficiency in ephrin-A1 inhibits breast cancer metastasis. F1000Research, 9, 217.

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Immunophenotyping of Hematopoietic Cells

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Cells were washed and resuspended at a concentration of 1 million cells per 100 μl in PBS + 0.5% BSA. Cells were stained with the indicated antibodies at the manufacturer’s suggested concentration for 20 minutes at 4°C, washed and acquired using a BD 4 Laser Fortessa flow cytometer. Antibodies: CD82 (BD, clone 423524), CD11b (BD, clone ICRF44), CD34 (BD, clone 581), CD38 (BD, clone HIT2).

Stengel K.R., Ellis J.D., Spielman C.L., Bomber M.L, & Hiebert S.W. (2020). Definition of a Small Core Transcriptional Circuit Regulated by AML1-ETO. Molecular cell, 81(3), 530-545.e5.

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Multiparameter Immune Cell Profiling

Cited 1 time

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Cells were stained with AlexaFlour700 viability dye. Drug and cytokine stimulations were performed similar to mass cytometry. Post-methanol permeabilization, cells were barcoded with serial dilutions of Pacific Blue and Pacific Orange dye as previously described.(30 (link)) Cells were stained with mAb cocktails (Supp. Table 2) for 30 minutes. Cells were resuspended prior to acquisition on the BD 4-laser Fortessa.

Sunthankar K.I., Jenkins M.T., Cote C.H., Patel S.B., Welner R.S, & Ferrell P.B. (2021). Isocitrate dehydrogenase mutations are associated with altered IL-1β responses in acute myeloid leukemia.. Leukemia, 36(4), 923-934.

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Multi-Parameter Flow Cytometry Protocol

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For traditional flow cytometry, 2 - 5 x10⁵ cells were plated, while for high parameter analysis using the Cytek Aurora 1 x 10⁶ cells from each site were used. Cells were washed with PBS and incubated with either near-infrared (traditional flow cytometry) or blue (Cytek Aurora) fixable live/dead stain (Life Technologies), as per the manufacturer’s instructions. Before incubation with human fc block (BD Pharmingen) cells were washed with FACS buffer (1% FCS, 2.5% HEPES, 1mM EDTA) and surface staining was performed at 4°C for 30 minutes using antibody panels as described in the key resources table. Surface staining was followed by washing with FACS buffer and fixation with 1% paraformaldehyde for 10 minutes. Labelled cells were acquired on a 4-laser BD Fortessa (traditional flow cytometry; BD Bioscience) or 5-laser Cytek Aurora flow cytometer (Cytek Bio).

Vijayakumar B., Boustani K., Ogger P.P., Papadaki A., Tonkin J., Orton C.M., Ghai P., Suveizdyte K., Hewitt R.J., Desai S.R., Devaraj A., Snelgrove R.J., Molyneaux P.L., Garner J.L., Peters J.E., Shah P.L., Lloyd C.M, & Harker J.A. (2022). Immuno-proteomic profiling reveals aberrant immune cell regulation in the airways of individuals with ongoing post-COVID-19 respiratory disease. Immunity, 55(3), 542-556.e5.

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Culturing Immune Cell Lines and Isolating PBMCs

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The following cell lines were cultured in RPMI 1640 (Irvine Scientific, USA) medium supplemented with 10% fetal bovine serum (FBS; Tissue Culture Biologicals [TCB], Long Beach, CA), 2 mM L-glutamine, 100 units/ml penicillin, 10 μg/ml streptomycin (Life Technologies, Grand Island, NY): Jurkat, a human T cell lymphoblast-like cell line (DSMZ, Braunschweig, Germany); and Raji, a Burkitt’s lymphoma cell line (ATCC, Rockville, MD). B16F10.9 (ATCC, Rockville, MD) cells were stably transfected with the plasmid pRG984/hCD20, a vector expressing human CD20. The resulting cell line (B16-CD20) was maintained in culture under media conditions described above along with puromycin selection (1 ug/ml). B16-CD20 cells were confirmed for expression of hCD20 by flow cytometry (4-laser BD Fortessa, BD Biosciences, San Jose, CA) using anti-human CD20 clone 2H7 (Biolegend, San Diego, CA). Data were analyzed using Cytobank software.
Human peripheral blood mononuclear cells (PBMC) were isolated from leukapheresis products obtained from normal healthy donors (New York Blood Center, NY, NY) using Ficoll-Paque, (GE Healthcare). Cynomolgus monkey PBMC were isolated from freshly drawn (within 3 hours) heparinized whole blood (Huntingdon Life Science, East Millstone, NJ) using Lympholyte-Mammal Cell Separation Media (Cedarlane, Burlington, Ontario, Canada)

Smith E.J., Olson K., Haber L.J., Varghese B., Duramad P., Tustian A.D., Oyejide A., Kirshner J.R., Canova L., Menon J., Principio J., MacDonald D., Kantrowitz J., Papadopoulos N., Stahl N., Yancopoulos G.D., Thurston G, & Davis S. (2015). A novel, native-format bispecific antibody triggering T-cell killing of B-cells is robustly active in mouse tumor models and cynomolgus monkeys. Scientific Reports, 5, 17943.

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T Cell Stimulation and Intracellular Cytokine Analysis

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To stimulate T cells, single cell suspensions were incubated for 5 hours at 37°C (5% CO₂) with 50 ngmL^-1 phorbol myrisate acetate (PMA) and 1 μgmL^-1 ionomycin (Biolegend, California) in the presence of Golgi-Stop (BD) for the last 4 hours. For cell surface staining, samples were blocked using 30 mL normal rat serum (StemCell Technologies) and incubated in the antibody cocktail for 20 minutes at 4°C in the dark. Intracellular cytokine staining was performed using Cytofix/Cytoperm (BD, New Jersey) according to manufacturer’s instructions. Flow cytometric analysis was performed using a 4-Laser Fortessa, 5-laser LSRII (BD) with FACSDiva software (BD), or a Cytek Aurora (Cytek, CA). Fluorescence-activated cell sorting (FACS) was performed on a FACS Aria III (BD). Analyses were performed using FlowJo (BD Biosciences). For all flow experiments, a live/dead stain (ThermoFisher) was used to only assess live cells. Antibodies used for flow cytometry or cell sorting are listed in Supplementary Table S1.

Jacobse J., Pilat J.M., Li J., Brown R.E., Kwag A., Buendia M.A., Choksi Y.A., Washington M.K., Williams C.S., Markham N.O., Short S.P, & Goettel J.A. (2024). Distinct roles for interleukin-23 receptor signaling in regulatory T cells in sporadic and inflammation-associated carcinogenesis. Frontiers in Oncology, 13, 1276743.

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Multiparametric Flow Cytometry Analysis

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Flow cytometry was performed on peripheral blood or BM single-cell suspensions after red blood cell lysis. All antibodies were from BioLegend: CD11b (clone M1/70), Gr1 (clone RB6-8C5), CD19 (clone 6D5), B220 (clone RA3-6B2), TCRβ (clone H57-597), c-Kit (clone 2B8), Sca-1 (clone D7), CD45.1 (clone A20), CD45.2 (clone 103). To assess progenitor population, mature cells were excluded by a lineage cocktail including anti-CD11b, –Gr-1, -B220, -CD19, -TCRβ, -CD8α (clone 53-6.7), -CD3 (clone 17A2), -TCRγ/δ (clone GL3), -CD11c (clone N418), -NK1.1 (clone PK136), and -Ter119 (all from BioLegend). Nonviable cells were excluded from analysis with Zombie Aqua Fixable Viability Dye (BioLegend), 7-amino-actinomycin D (BioLegend), or DAPI (MilliporeSigma). Flow cytometric analysis was performed using a 4-laser Fortessa (BD). FlowJo (Tree Star) was used for data analysis.

Grill S., Padmanaban S., Friedman A., Perkey E., Allen F., Tesmer V.M., Chase J., Khoriaty R., Keegan C.E., Maillard I, & Nandakumar J. (2021). TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis. JCI Insight, 6(9), e138059.

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Multicolor Flow Cytometry Immunophenotyping

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Fluorochrome-coupled anti-mouse CD4 (GK1.5), CD5 (53–7.3), CD25 (PC61.5), CD44 (1M7), CD45 (30F11), CD45.1 (A20), CD45.2 (104), CD8α (53−6.7), CD8β (REA793 or H35–17.2), CD122 (TM-bl), TCRβ (H57–597), TCRγδ (eBioGL3), Ki69 (solA15), PD-1 (J43.1), and isotype controls were purchased from Thermofisher, BD Biosciences or Tonbo. Annexin V and 7AAD were purchased from BD Biosciences. All staining samples were acquired using BD FACS Canto II, 4-Laser Fortessa, or 5-Laser LSR II Flow cytometers (BD Biosciences) and data was analyzed using FlowJo software (Tree Star). Cell staining was performed following conventional techniques. Manufacturer’s instructions were followed for Annexin V staining; briefly, early apoptotic cells were considered as annexin V⁺7AAD^neg, late apoptotic cells or undergoing necrosis as annexin V⁺7AAD⁺, and necrotic cells as annexin V^-7AAD⁺. FACS sorting was performed using a FACSAria III at the Flow Cytometry Shared Resource at VUMC.

Nazmi A., Greer M.J., Hoek K.L., Piazuelo M.B., Weitkamp J.H, & Olivares-Villagómez D. (2020). Osteopontin and iCD8α cells promote intestinal intraepithelial lymphocyte homeostasis. Journal of immunology (Baltimore, Md. : 1950), 204(7), 1968-1981.

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Multiparameter Flow Cytometry Analysis

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The following antibodies were from Biolegend: anti-CD4 (clone GK1.5); CD8α (clone 53-6.7); CD44 (clone IM7); CD25 (clone PC61); CD45.1 (clone A20); CD45.2 (clone 104); Thy1.1 (clone OX-7); Thy1.2 (clone 30-H12); H-2K^b (clone AF6-88.5); H-2K^d (clone SF1-1.1); IFNγ (clone XMG1.2); IL-17A (clone TC11-18H10.1); pan-CD43 (clone S11), core-2 O-glycosylation CD43 (clone 1B11); KLRG1 (clone 2F1); CD127/IL7Rα (clone A7R34). Anti-FoxP3 (clone FJK-16s) was from eBioscience. Anti-Vβ13 (clone MR12-3) was from BD Biosciences. SIINFEKL peptide loading on H-2K^b MHC and conjugation to PE was done as previously described (43 (link)). Non-viable cells were excluded from analysis with Zombie Aqua Fixable Viability Dye (Biolegend), or DAPI (Sigma-Aldrich). For intranuclear staining, FoxP3/Transcription Factor Staining Buffer (eBioscience) was used per manufacturer’s protocol. Assessment of intracellular T cell cytokine production after short ex vivo stimulus was previously described (10 (link)). Flow cytometric analysis was performed using a 4-laser Fortessa (BD). Sorting of T cells was performed using a 4-laser FACSAria II/III (BD). Alloreactive 4C T cells were defined as Thy1.1⁺ CD4⁺. Naïve OT-I CD8⁺ T cells (CD8⁺CD44^lo) and day 3 OT-I effector CD8⁺ T cells (CD8⁺CD45.2⁺CD44^hi) were sorted on a BD FACSARIA II.

Perkey E., De Sousa D.M., Carrington L., Chung J., Dils A., Granadier D., Koch U., Radtke F., Ludewig B., Blazar B.R., Siebel C.W., Brennan T.V., Nolz J., Labrecque N, & Maillard I. (2020). GCNT1-mediated O-glycosylation of the sialomucin CD43 is a sensitive indicator of Notch signaling in activated T cells. Journal of immunology (Baltimore, Md. : 1950), 204(6), 1674-1688.

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Multiparametric Cell Surface and Intracellular Staining

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For cell surface staining, cells were incubated in the antibody cocktail for 20 minutes at 4°C in the dark. Samples were blocked using 30 μL normal rat serum (StemCell Technologies). Intracellular cytokine staining was performed using Cytofix/Cytoperm (BD) and intranuclear stain was performed using the FOXP3 staining buffer set (eBioscience, San Diego, California), both according to manufacturer’s instructions. Filipin-III was stained after overnight fixation of cells. Annexin-V staining was performed following the manufacturers’ instructions (ThermoFisher Scientific) by staining for 15 minutes at room temperature using Ca²⁺-containing Annexin-V buffer (diluted from 5X stock to 1X with ultrapure water). Cells were washed in room temperature also using Annexin V buffer prior to analysis. Flow cytometric analysis was performed using a 4-Laser Fortessa or 5-laser LSRII (BD) with FACSDiva software (BD). Fluorescence-activated cell sorting (FACS) was performed on a FACS Aria III (BD). Analyses were performed using FlowJo (BD Biosciences). For all flow experiments, a live/dead stain (ThermoFisher) was used to only assess live cells. Antibodies used for flow cytometry or cell sorting are listed in Table S2.

Jacobse J., Brown R.E., Li J., Pilat J.M., Pham L., Short S.P., Peek C.T., Rolong A., Washington M.K., Martinez-Barricarte R., Byndloss M.X., Shelton C., Markle J.G., Latour Y.L., Allaman M.M., Cassat J.E., Wilson K.T., Choksi Y.A., Williams C.S., Lau K.S., Flynn C.R., Casanova J.L., Rings E.H., Samsom J.N, & Goettel J.A. (2023). Interleukin-23 receptor signaling impairs the stability and function of colonic regulatory T cells. Cell reports, 42(2), 112128.

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