Facscanto flow cytometer

Manufactured by FlowJo

The FACSCanto flow cytometer is a high-performance instrument used for the analysis of cells and other particles in a fluid sample. It is capable of detecting and quantifying multiple parameters of individual cells, such as size, granularity, and the expression of specific proteins or markers on the cell surface. The FACSCanto is designed to provide researchers with accurate and reliable data for a wide range of applications, including immunology, hematology, and cell biology.

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Lab products found in correlation

Countess 2, by Thermo Fisher Scientific (3 mentions) Anti mouse cd11b, by BioLegend (1 mentions) Anti mouse gr 1, by BioLegend (1 mentions) Anti mouse cd3, by BioLegend (1 mentions) Collagenase type 4, by Merck Group (1 mentions) Dnase 1, by Merck Group (1 mentions) C12fdg, by Thermo Fisher Scientific (1 mentions) Red fluorescent beads, by Bangs Laboratories (1 mentions) Pe conjugated cd44, by BD (1 mentions) Cd24 fitc, by BD (1 mentions) 7 aad, by BD (1 mentions) Cd44 pe, by BD (1 mentions) Facsdiva software version 7, by BD (1 mentions) Lsr 2, by BD (1 mentions) Click it edu flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions) Cuprizone, by Merck Group (1 mentions) Cd106, by BD (1 mentions) Igg2b, by Thermo Fisher Scientific (1 mentions) Igg1κ, by Thermo Fisher Scientific (1 mentions) Cd105, by BD (1 mentions)

17 protocols using facscanto flow cytometer

Cell Cycle Analysis by Flow Cytometry

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Cells were fixed in ice-cold ethanol, washed in PBS-1% FBS and treated with 2 M HCl for 20 min at 37 °C, and subsequently with 50 μg/ml Propidium Iodide/200 μg/ml RNase A in PBS. Analysis was performed using a Becton Dickinson FACS Canto Flow Cytometer and FlowJo software.
To discriminate between cells in G2 and M, cells were fixed in 0.5% (w/v) para-formaldehyde for 20 min at 37 °C and incubated with 100 μl of 10 μg/ml anti-phospho-(S10)-histone H3 antibody for 1 h, followed by washing in PBS-1% FBS and incubation with 12.5 μg/ml FITC-labeled goat anti-rabbit IgG for 30 min. Cells were subsequently washed, stained with propidium iodide and analyzed as above.

Smith L., Farzan R., Ali S., Buluwela L., Saurin A.T, & Meek D.W. (2017). The responses of cancer cells to PLK1 inhibitors reveal a novel protective role for p53 in maintaining centrosome separation. Scientific Reports, 7, 16115.

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Isolation and Characterization of Lung Cells

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IR-61 (2 mg kg⁻¹) was injected i.v. into mice. 24 h later, the lungs were minced and chopped to approximately 10 mm pieces. Then the tissues were digested in DMEM medium containing 2 mg mL⁻¹ of type IV collagenase (#C5138, Sigma), 0.125 mg mL⁻¹ DNase I (#C5025, Sigma) for 30 min at 37 °C and subsequently the resulting suspension passed through a 70 μm cell strainer. After red cell lysis, the cells were blocked with 1% of rat anti mouse CD16/CD32 (#553141, BD Biosciences, RRID: AB_394656) and then stain with anti-mouse CD3 (#100218, Biolegend, RRID: AB_1595492), anti-mouse CD11b (#101205, Biolegend, RRID: AB_312788), anti-mouse Gr1 (#108433, Biolegend, RRID:AB_10900232), anti-mouse CD19 (#115520, Biolegend, RRID: AB_313655), anti-mouse F4/80 (#123116, Biolegend, RRID: AB_893481), and anti-mouse CD11c (#117308, Biolegend, RRID: AB_313777) for 30 min at 4 °C. The acquisition was performed on a FACSCanto flow cytometer with FACSDiva software, and subsequent analysis was performed using FlowJo V10.1.

Wang Y., Tang B., Li H., Zheng J., Zhang C., Yang Z., Tan X., Luo P., Ma L., Wang Y., Long L., Chen Z., Xiao Z., Ma L., Zhou J., Wang Y, & Shi C. (2023). A small-molecule inhibitor of Keap1–Nrf2 interaction attenuates sepsis by selectively augmenting the antibacterial defence of macrophages at infection sites. eBioMedicine, 90, 104480.

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Measuring Phagolysosomal β-Galactosidase Activity

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To assess β-galactosidase activity in phagolysosomes, either S. Typhimurium (SL1344) or red fluorescent beads (Bangs Laboratories) were coated with 5-dodecanoylaminofluorescein Di-β-d-Galactopyranoside (C₁₂FDG, Life Technologies) for 60 min at 37 °C in NaHCO₃ buffer (pH 9.6). Infection was carried out at MOI of 10, as described above, or 100 beads per cell were added to BMDMs and incubated for 10 min at RT and then 10 min at 37 °C, followed by washes with RPMI to remove extracellular beads. After 10 min or 0.5, 1, 2, or 4 h, the cells were washed with cold PBS and fixed with 1% formaldehyde for 10 min. After intensive washing, the cells were collected in 1% BSA/PBS and samples were acquired on a FACS Canto flow cytometer (26 (link)), and the data were analyzed using FlowJo software. The mean fluorescence intensities (MFIs) of C₁₂FDG was normalized to the red MFI of the beads for every sample.

Fischer J., Gutièrrez S., Ganesan R., Calabrese C., Ranjan R., Cildir G., Judith Hos N., Rybniker J., Wolke M., Fries J.W., Tergaonkar V., Plum G., Antebi A, & Robinson N. (2019). Leptin signaling impairs macrophage defenses against Salmonella Typhimurium. Proceedings of the National Academy of Sciences of the United States of America, 116(33), 16551-16560.

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Isolation and Characterization of Cancer Stem Cells

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A single-cell suspension was double-stained with PE-conjugated CD44 (BD Biosciences) and FITC-conjugated CD49f (BD Biosciences) antibodies to sort out the CD44+/CD49f+ and CD44-/CD49f- populations as described previously [4 (link)]. For knockdown experiments, cells were treated with siBMI1 (10nM) for 96 hours, re-suspended in re-suspension buffer (2% FBS in 1x PBS), and stained with CD44-PE (1:20; BD Biosciences) and CD24-FITC (1:5; BD Biosciences) for 30 minutes at 4°C. Cells were washed with re-suspension buffer and stained with 7-AAD (1:20; BD Biosciences). Data was acquired using a FACSCanto flow cytometer and analyzed using FlowJo software.

Davies A.H., Reipas K.M., Pambid M.R., Berns R., Stratford A.L., Fotovati A., Firmino N., Astanehe A., Hu K., Maxwell C., Mills G.B, & Dunn S.E. (2014). YB-1 transforms human mammary epithelial cells through chromatin remodeling leading to the development of basal-like breast cancer. Stem cells (Dayton, Ohio), 32(6), 1437-1450.

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Erythroblast Surface Marker Analysis

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Bone marrow erythroblasts from Patient 2 were analyzed at three consecutive timepoints for surface expression of glycophorin A (GPA), α4 integrin and band 3 as previously described^{29 (link)}.
The following flurophore-conjugated antibodies were used: anti-human APC-α4 integrin (clone MZ18–24A9, Miltenyi Biotec, #130–124-008, 1:100), anti-human PE-glycophorin A (GPA or CD235a; clone JC159, Dako Cytomation, #R7078, 1:100). Mouse monoclonal antibodies against the extracellular regions of human band 3 were generated by the New York Blood Center (1:100).
Data was collected using the FACSDiva software version 7.0 (BD) on a FACSCanto™ flow cytometer and analyzed using the FlowJo software version 7.6.5.

Boulad F., Maggio A., Wang X., Moi P., Acuto S., Kogel F., Takpradit C., Prockop S., Mansilla-Soto J., Cabriolu A., Odak A., Qu J., Thummar K., Du F., Shen L., Raso S., Barone R., Di Maggio R., Pitrolo L., Giambona A., Mingoia M., Everett J.K., Hokama P., Roche A.M., Cantu V.A., Adhikari H., Reddy S., Bouhassira E., Mohandas N., Bushman F.D., Rivière I, & Sadelain M. (2022). A phase 1 trial of lentiviral globin gene therapy with reduced intensity conditioning in adults with transfusion dependent β-thalassemia. Nature medicine, 28(1), 63-70.

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Neutrophil Reactive Oxygen Detection

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Cocultured neutrophils were harvested and washed with PBS, and incubated with 10 µM DCFH-DA for 30 min at 37°C. After the incubation period, the fluorescence was analyzed using flow cytometry. Results are presented as arbitrary units of fluorescence. All experiments were conducted with a FACS Canto Flow Cytometer and analyzed using the Flow Jo software (version 9.1). Data from 10,000 cells were obtained, and only the morphologically viable cells were considered in the analysis.

Drewes C.C., Alves A.D., Hebeda C.B., Copetti I., Sandri S., Uchiyama M.K., Araki K., Guterres S.S., Pohlmann A.R, & Farsky S.H. (2017). Role of poly(ε-caprolactone) lipid-core nanocapsules on melanoma–neutrophil crosstalk. International Journal of Nanomedicine, 12, 7153-7163.

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T Cell Proliferation Assay with CFSE

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Plates (24 well) were coated with anti-CD3 (1 μg/ml) or anti-CD3 (1 μg/ml) and anti-CD28 (10 μg/ml) for overnight at 4°C. Single cell suspensions of thymocytes were washed with PBS without Ca²⁺ and Mg²⁺ twice, then resuspended at 2 × 10⁷ cells/ml. A CFSE solution was added to the cells to a final concentration of 2.5 μM for 8 min at room temperature. After 8 min, fetal calf serum was directly added to the CFSE labeled cells and incubated for 1 min. Cells were washed twice with 5 min interval. 2,000,000 of CFSE labeled cells were added to pre-coated plates. After 72 hr, cells were harvested and stained for surface markers as well as DAPI (4′,6-diamidino-2-phenylindole). Data were collected on the BD LSRII or FACSCanto flow cytometer and analyzed using FlowJo software.

Phee H., Au-Yeung B.B., Pryshchep O., O'Hagan K.L., Fairbairn S.G., Radu M., Kosoff R., Mollenauer M., Cheng D., Chernoff J, & Weiss A. (2014). Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation. eLife, 3, e02270.

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Flow Cytometry-based Cell Sorting for Osteoclastogenesis

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2×10⁶ BM or cultured cells were stained with APC-Ly6C, PEcy7-CD11b, PE-Gr1 and PECy5-F4/80 antibodies for 30 min. Data were acquired using a FACScanto flow cytometer and analyzed using FlowJo software, as described previously [26 (link), 30 (link)]. For cell sorting, cultured OCPs from WT mice were stained with the above fluorescent-conjugated antibodies to confirm that each cell population was similar to those generated in our regular culture procedure, and the cells were then sorted by a Statler sorter to collect Ly6C⁺Gr1^-, Ly6C⁺Gr1⁺, Ly6C^-Gr1^- and Ly6C^-Gr1⁺ cells separately. The sorted cells were reanalyzed to ensure their purity (≥98%) and used for osteoclastogenesis assays, as we described previously [26 (link)].

Zhao Z., Hou X., Yin X., Li Y., Duan R., Boyce B.F, & Yao Z. (2015). TNF Induction of NF-κB RelB Enhances RANKL-Induced Osteoclastogenesis by Promoting Inflammatory Macrophage Differentiation but also Limits It through Suppression of NFATc1 Expression. PLoS ONE, 10(8), e0135728.

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Detecting Antigen-Specific T Cell Responses

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Total memory CD8 T cells were detected using the surrogate activation markers CD8 and CD11a as previously described40 (link). Endogenous GP₃₃-specific memory CD8 T cells were detected with MHC class I GP₃₃ tetramers, and memory P14 cells were gated on CD8/Thy1.1+ cells. Ex vivo activation status following infection was determined by incubating splenocytes at 37 °C for 1 h in the presence of BrefeldinA (BFA) followed by intracellular staining with antibodies (Abs) against either IFN-γ or GrB or extracellular staining with Abs against either CD25 or CD69. All cells for flow cytometry were analyzed using a FACSCanto flow cytometer and analyzed using FlowJo software.

Martin M.D, & Badovinac V.P. (2015). Antigen-dependent and –independent contributions to primary memory CD8 T cell activation and protection following infection. Scientific Reports, 5, 18022.

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Measuring Cell Proliferation with EdU Assay

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To measure the proliferative ability of the TM4 and human Sertoli cells, DNA synthesis was monitored using 5-ethynyl-2´-deoxyuridine (EdU) staining. EdU, a thymidine analog nucleoside, is incorporated into DNA and is correlated with DNA synthesis activity (Buck et al., 2008 (link)). Cells were seeded in 6-well plates for 24 h and then exposed to CBD or its metabolites for 24 h. A Click-iT^® EdU Flow Cytometry Assay kit (Thermo Fisher Scientific) was used to supply 10 μM EdU to the cells for 2 h before DNA synthesis detection, which was based on the copper-catalyzed reaction between the azide moiety in Alexa Fluor^® 488 dye and the alkyne group in EdU. Before staining, the number of cells was adjusted to 10⁶ cells/sample. Fixation, permeabilization, and Click-iT^® reaction steps were performed following the manufacturer’s protocol. The FACSCanto^™ flow cytometer with FACSDiva^™ software was used to detect Alexa Fluor^® 488 signals and FlowJo^® software was used to determine the percentage of cells in the S phase.

Alland D., Kramnik I., Weisbrod T.R., Otsubo L., Cerny R., Miller L.P., Jacobs WR J.r, & Bloom B.R. (1998). Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): The effect of isoniazid on gene expression in Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America, 95(22), 13227-13232.

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