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Fortessa cytofluorimeter

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The Fortessa cytofluorimeter is a flow cytometry instrument designed for high-performance cell analysis. It is capable of detecting and analyzing multiple fluorescent signals from cells or particles in a fluid stream. The core function of the Fortessa cytofluorimeter is to provide researchers with a tool for quantitative and qualitative analysis of various cellular characteristics, such as size, granularity, and the expression of specific markers on the cell surface or within the cell.

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Lab products found in correlation

Facsdiva version 8, by BD (1 mentions) Sp6800, by Sony (1 mentions) Collagenase type 4, by Merck Group (1 mentions)

3 protocols using fortessa cytofluorimeter

1

Multiparametric Flow Cytometry of Immune Cells

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Cells were harvested from spleen or draining lymph nodes were dispersed into PBS through a 40 micron cell strainer using the back of a 1 mL syringe plunger. Cell preparations were subjected to hypotonic lysis to remove erythrocytes, stained and analyzed using a Fortessa cytofluorimeter (BD). Flow cytometry fluorescent dye–conjugated antibodies to the following proteins were purchased from Biolegend (B220-PacificBlue [clone RA3-6B2], CD107a-Fitc [1D4B], CD11b-AlexaFluorophore 488 [M1/70], CD11c-APC [N418], CD11c-PE/Cy7 [N418], CD25-AlexaFluorophore 488 [PC61], CD45-Bv711 [30-F11], CD4-APC [RM4-5], CD4-PacificBlue [RM4-5], CD69-PE [H1.2F3], CD8-Bv650 [53-6.7], FoxP3-PE [MF-14], Gr1-PE [RB6-8C5], Gr1-PE/Cy7 [RB6-8C5], MHC I-A/I-E-Bv510 [M5/114.15.2], NK1.1-FITC [PK136], NK1.1-PE/Cy7 [PK136], PD-1-PE/Cy7 [29F.1A12], Tim3-APC [RMT3–23], PD-L1-APC [10F.9G2]), Affymetrix (CD19-PE [MB19-1]).
Dougan M., Ingram J.R., Jeong H.J., Mosaheb M.M., Bruck P.T., Ali L., Pishesha N., Blomberg O., Tyler P.M., Servos M.M., Rashidian M., Nguyen Q.D., von Andrian U.H., Ploegh H.L, & Dougan S.K. (2018). Targeting cytokine therapy to the pancreatic tumor microenvironment using PD-L1 specific VHHs. Cancer immunology research, 6(4), 389-401.
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2

Competitive Fitness Assay with BTZ

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Next, 20 000 cells were infected at a multiplicity of infection of ∼1.2 to ensure 100% transduction efficiency with either virus particles of NLS-mCherry LacZ-sgRNA or NLS-BFP GOI-sgRNA. After transduction (96 hours), mCherry- and BFP-expressing cells were mixed 1:1 and plated with or without BTZ (4 nM) in a 12-well format. The cells were subcultured when near-confluency was reached, and BTZ containing medium was replaced every 3 days. Cells were imaged for BFP and mCherry signal the day of initial plating (t = 0) and on days 4, 8, 12, and 16. Data were acquired using the fluorescence-activated cell sorter (FACS) Fortessa cytofluorimeter and processed with FACSDiva version 8.0 software (BD Biosciences).
Findlay S., Nair R., Merrill R.A., Kaiser Z., Cajelot A., Aryanpour Z., Heath J., St-Louis C., Papadopoli D., Topisirovic I., St-Pierre J., Sebag M., Kesarwala A.H., Hulea L., Taylor E.B., Shanmugam M, & Orthwein A. (2023). The mitochondrial pyruvate carrier complex potentiates the efficacy of proteasome inhibitors in multiple myeloma. Blood Advances, 7(14), 3485-3500.
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3

Pancreatic Tumor Dissociation and Flow Cytometry

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Pancreatic tumors were excised, weighed, minced and incubated in RPMI-1640 containing collagenase type IV (Sigma) and anti-trypsin at 37°C for 1 hour. Tumors were filtered through a 40 micron cell strainer, washed with PBS, and centrifuged. The resulting cell pellet containing tumor debris and infiltrating immune cells was resuspended in FACS buffer (PBS with 2% fetal calf serum) and stained with a master mix of antibodies. Cells were incubated with staining mix for 30 minutes at 4°C, washed once in PBS, and resuspended in 1% formalin prior to analysis on either a spectral flow cytometer (Sony SP6800) or a Fortessa cytofluorimeter (BD). Flow cytometry antibodies used in this study were purchased from BioLegend and used at 1:200 dilution (αCD45[30-F11], αCD4[RM4–5], αCD8[53–6.7], αNK1.1[PK136], αCD103[2E7], Ly6C[1A8], I-A/I-E[M5/114.5.2], F4/80[BM8], SigF[E50–2440], αCD11b[M1170] αCD11c[N418], αGR1[RB6–8C5], H2-Kb[AF6–88.5]). For one experiment, we used an ImageStreamx MkII Imaging flow cytometer (IFC) (Amnis Corporation) and IDEAS™ersion 6.2.64.0 software was used for compensation and data analysis.
Roehle K., Qiang L., Ventre K., Heid D., Ali L.R., Lenehan P., Heckler M., Crowley S.J., Stump C.T., Ro G., Godicelj A., Bhuiyan A.M., Yang A., del Rey M.Q., Biary T., Luoma A.M., Bruck P.T., Tegethoff J.F., Nopper S.L., Li J., Byrne K.T., Pelletier M., Wucherpfennig K.W., Stanger B.Z., Akin J.J., Mancias J.D., Agudo J., Dougan M, & Dougan S.K. (2021). cIAP1/2 antagonism eliminates MHC class I negative tumors through T cell-dependent reprogramming of mononuclear phagocytes. Science translational medicine, 13(594), eabf5058.
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