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Facsfusion

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FACSFusion is a flow cytometry instrument designed for sensitive and precise cell analysis. It enables the detection and quantification of various cellular parameters, including size, granularity, and fluorescence intensity.

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Lab products found in correlation

Facsaria 2, by BD (8 mentions) Facsaria, by BD (5 mentions) Lsrfortessa, by BD (5 mentions) Facsaria 3, by BD (4 mentions) Collagenase type 1, by Merck Group (3 mentions) Anti cd45.2 104, by BioLegend (3 mentions) Anti cd31 390, by BioLegend (3 mentions) Collagenase xi, by Merck Group (3 mentions) Facsaria iiiu, by BD (3 mentions) Hyaluronidase, by Merck Group (3 mentions) Tryple express, by Thermo Fisher Scientific (3 mentions) Lsrfortessa x 20, by BD (3 mentions) Gentlemacs dissociator, by Miltenyi Biotec (3 mentions) Cd11b, by Thermo Fisher Scientific (3 mentions) Anti cd11b m1 70, by BioLegend (2 mentions) Anti cd68 fa 11, by BioLegend (2 mentions) Pe anti mcd47 miap301, by BioLegend (2 mentions) Accuri c6, by BD (2 mentions) Nextseq 500, by Illumina (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)

Close spelling variants of this product

BD FACSFusion cytometer (5 citations) BD FACSFUSION system (2 citations)

64 protocols using facsfusion

1

Isolation and Analysis of Cell Populations

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Cells were isolated 72 h after injection with LNPs unless otherwise noted. Mice were perfused with 20 mL of 1× PBS through the right atrium. Tissues were finely cut and then placed in a digestive enzyme solution with collagenase type I (Sigma-Aldrich), collagenase XI (Sigma-Aldrich), and hyaluronidase (Sigma-Aldrich) at 37 °C at 550 rpm for 45 min. The digestive enzyme for heart and spleen included collagenase IV (32 (link), 37 , 45 (link)). Cell suspension was filtered through 70-μm mesh and red blood cells were lysed. Cells were stained to identify specific cell populations and sorted using the BD FacsFusion and BD Facs Aria IIIu cell sorters in the Georgia Institute of Technology Cellular Analysis Core. For in vitro experiments, BD Accuri C6 and BD FacsFusion were used. The antibody clones used were anti-CD31 (390; BioLegend), anti-CD45.2 (104; BioLegend), and anti-CD102 (3C4; BioLegend). PE anti-mCD47 (miap301; BioLegend) was used for tdTomato compensation. Representative flow gates are located in SI Appendix, Fig. S8. PBS-injected Ai14 mice were used to gate tdTomato populations for i.v. administration, while contralateral limbs were used to gate for intramuscular experiments.
Sago C.D., Lokugamage M.P., Paunovska K., Vanover D.A., Monaco C.M., Shah N.N., Gamboa Castro M., Anderson S.E., Rudoltz T.G., Lando G.N., Munnilal Tiwari P., Kirschman J.L., Willett N., Jang Y.C., Santangelo P.J., Bryksin A.V, & Dahlman J.E. (2018). High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing. Proceedings of the National Academy of Sciences of the United States of America, 115(42), E9944-E9952.
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2

Immune Cell Profiling by FACS

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Cells were stained to identify specific cell populations and sorted using the BD FacsFusion and BD Facs Aria IIIu cell sorters in the Georgia Institute of Technology cellular analysis core. Antibodies used for staining were CD31 (clone 390, BioLegend), CD45.2 (clone 104, BioLegend), and CD11b (clone M1/70, BioLegend).
We defined cell populations in the following manner: macrophages (CD31−CD45+CD11b+), heart and lung endothelial cells (CD31+CD45−), bone marrow endothelial cells (CD31+) (S3A–C).
Paunovska K., Sago C.D., Monaco C.M., Hudson W.H., Castro M.G., Rudoltz T.G., Kalathoor S., Vanover D.A., Santangelo P.J., Ahmed R., Bryksin A.V, & Dahlman J.E. (2018). A direct comparison of in vitro and in vivo nucleic acid delivery mediated by hundreds of nanoparticles reveals a weak correlation. Nano letters, 18(3), 2148-2157.
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3

Multimodal Analysis of Immune Cells

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Cells were isolated 72 hours after injection with LNPs unless otherwise noted. Mice were perfused with 20 mL of 1X PBS through the right atrium. Liver and lung tissues were finely minced, and then placed in a digestive enzyme solution with Collagenase Type I (Sigma Aldrich), Collagenase XI (Sigma Aldrich) and Hyaluronidase (Sigma Aldrich) at 37 ºC at 550 rpm for 45 minutes4,5. The spleen, bone marrow and thymus tissues were placed in 1X PBS solution. Cell suspension was filtered through 70μm mesh and red blood cells were lysed. Cells were stained to identify specific cell populations and sorted using the BD FacsFusion and BD Facs Aria IIIu cell sorters in the Georgia Institute of Technology Cellular Analysis Core. The antibody clones used were the following: anti‐CD31 (390, BioLegend), anti‐CD45.2 (104, BioLegend), anti‐CD68 (FA11, Biolegend), anti‐CD3 (17A2, Biolegend), anti‐CD19 (6D5, Biolegend), anti‐CD11b (M1/70, Biolegend), anit‐CD11c (N418, Biolegend), anti‐CD4 (GK1.5, Biolegend), anti‐CD8a (53‐6.7, Biolegend), anti‐CD34 (SA376A4, Biolegend), anti‐CD25 (3C7, Biolegend), anti‐CD326 (G8.8, Biolegend), and PE anti‐mCD47 (miap301, BioLegend). Representative flow gates are located in Supplementary Figure 3. PBS‐injected Ai14 mice were used to gate tdTomato populations for intravenous administration.
Gan Z., Lokugamage M.P., Hatit M.Z., Loughrey D., Paunovska K., Sato M., Cristian A, & Dahlman J.E. (2020). Nanoparticles containing constrained phospholipids deliver mRNA to liver immune cells in vivo without targeting ligands. Bioengineering & Translational Medicine, 5(3), e10161.
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4

Cell Cycle Sorting and siRNA Transfection

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Stable cell lines have been generated as in [12 (link)]. Cells were incubated with Hoechst 33342 (ThermoFisher Scientific H3570) at 1/1000 directly into the media for 1h at 37 degrees. Cells were then trypsinized and resuspended in media containing Hoechst at 1/1000. Cells were sorted with a BD FACS Fusion using the laser BV421-A into Qiazol, based on DNA content. The gates for the sorting were set such as the G1 phase was collected from the bottom left of the first peak to the middle of the right slop, the G2 phase was collected from the bottom left of the second peak to the bottom right of that same peak. The ES and LS phase were equally divided in between G1 and G2 phases. Figure 1A presents the gating.
The siRNA were transfected using Lipofectamine 3000 (ThermoFisher Scientific) according to the supplier recommendations. The siRNAs were purchased from ThermoFisher Scientific (Silencer Select): KDM4A (s18635, s18636, s18637), FUS/TLS (s5401, s5402), hnRNPUL1 (s21883, s21884).
Van Rechem C., Ji F., Mishra S., Chakraborty D., Murphy S.E., Dillingham M.E., Sadreyev R.I, & Whetstine J.R. (2020). The lysine demethylase KDM4A controls the cell-cycle expression of replicative canonical histone genes. Biochimica et biophysica acta. Gene regulatory mechanisms, 1863(10), 194624.
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5

Stable Cell Line Generation via FACS

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To create monoclonal stable cell lines, polyclonal cells were single cell sorted in a 96-well plate by fluorescence-activated cell sorting (FACS) using BD FACS Aria or BD FACS Fusion. Polyclonal cells were washed once with DPBS, collected by using Cellstripper (Corning). In the case of non-GFP tagged expression construct (5HT2A-APEX2), cells were incubated with Alexa 647-conjugated M1-anti-FLAG antibody (1:1000 dilution) at 4 °C for 40 min. After spinning down, cells were washed with ice-cold PBS and resuspended in 1mL of sample buffer (PBS/1mM EDTA/25 mM HEPES/1% FBS) for sorting. 24 clones were selected and expanded in a 24-well plate for characterization by flow cytometry using the Attune NxT Flow Cytometer. The cellular morphology was first gated based on an FSC-A vs. SSC-A (forward scatter - area vs. side scatter - area) plot. Next, gating was generated based on the FSC-A vs. FSC-H (area vs. height) plot to select singlets. These singlets were further analyzed to identify the cells expressing 5HT2A or the localization domains based on Alexa 647 or GFP fluorescence signal, respectively. The data was processed in FlowJo.
Zhong X., Li Q., Polacco B.J., Patil T., DiBerto J.F., Vartak R., Xu J., Marley A., Foussard H., Roth B.L., Eckhardt M., Von Zastrow M., Krogan N.J, & Hüttenhain R. (2023). An automated proximity proteomics pipeline for subcellular proteome and protein interaction mapping. bioRxiv.
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6

Antigen-Specific Splenocyte Profiling

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Splenocytes isolated from healthy controls and EAE mice on day 12 at peak of disease were seeded at 3×106 cells/mL (3×106 cells/well) in a 12-well plate and cultured in the presence or absence of 25 μM PLP antigen challenge plus treatment (dosed at 25 uM PLP) for 72 hours. Cells were then stained with PE/Cy7 anti-mouse CD3, AlexaFluor647 anti-mouse CD19, Pacific Blue anti-mouse CD11c, PerCP anti-mouse B220, PE anti-mouse CD86, and FITC anti-mouse CD80 per the manufacturer guidelines and resuspended in serum-free clear RPMI. Marker expression was evaluated by flow cytometry (BD FACSFusion).
Hartwell B.L., Pickens C.J., Leon M., Northrup L., Christopher M., Griffin J.D., Martinez-Becerra F, & Berkland C. (2018). Soluble antigen arrays disarm antigen-specific B cells to promote lasting immune tolerance in experimental autoimmune encephalomyelitis. Journal of autoimmunity, 93, 76-88.
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7

Isolation and Flow Cytometry Analysis of Tissue-Specific Cell Populations

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Cells were isolated 24 (for screens) or 96 (for in vivo gene editing) hours after injection with LNPs unless otherwise noted. Mice were perfused with 20 mL of 1X PBS through the right atrium. Tissues were finely cut, and then placed in a digestive enzyme solution with Collagenase Type I (Sigma Aldrich), Collagenase XI (Sigma Aldrich) and Hyaluronidase (Sigma Aldrich) at 37 ºC at 550 rpm for 45 minutes. The digestive enzyme for heart and spleen included Collagenase IV7 , 56 , 58 (link). Cell suspension was filtered through 70μm mesh and red blood cells were lysed. Cells were stained to identify specific cell populations and sorted using the BD FacsFusion and BD Facs Aria IIIu cell sorters in the Georgia Institute of Technology Cellular Analysis Core. For in vitro flow cytometry experiments, a BD Accuri C6 was used in the Georgia Institute of Technology Cellular Analysis Core. The antibody clones used were: anti-CD31 (390, BioLegend), anti-CD45.2 (104, BioLegend), anti-CD68 (FA-11, Biolegend), and anti-CD11b (M1/70, Biolegend). Representative flow gates are located in Supplementary Figure 1.
Sago C.D., Lokugamage M.P., Lando G.N., Djeddar N., Shah N.N., Syed C., Bryksin A.V, & Dahlman J.E. (2018). Modifying a commonly expressed endocytic receptor retargets nanoparticles in vivo. Nano letters, 18(12), 7590-7600.
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8

Chromatin Immunoprecipitation Sequencing Protocol

Cited 2 times
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Cells were incubated with Hoechst 33342 (ThermoFisher Scientific H3570) at 1/1000 directly into the media for 1h at 37°C degrees. Cells were then trypsinized and resuspended in media containing Hoechst at 1/1000 before crosslinking with 1% formaldehyde for 13min at 37°C degrees and quenching with 0.125M glycine. Cells were washed with 1X PBS and resuspended in media containing Hoechst (1/1000). Cells were sorted with a BD FACS Fusion using the laser BV421-A based on DNA content. For siKDM5A ChIP, cells were harvested as previously described (10 (link),18 (link)). Sonication of chromatin was done with the Qsonica Q800R2 system (Qsonica). 0.5–10ug of chromatin were used based on DNA content (nanodrop concentrations) with the following antibodies: H3K4me1 (Abcam ab8895), H3K4me2 (Abcam ab32356), H3K4me3 (Millipore 07–473), H3K9me1 (Abcam ab8896–100), H3K9me2 (Abcam ab1220), H3K9me3 (Abcam ab8898). ChIP sequencing libraries were prepped using the TruSeq ChIP Sample Preparation kit (Illumina). Libraries were single-end sequenced (75 cycles) using a NextSeq500 (Illumina).
Clarke T.L., Tang R., Chakraborty D., Rechem C.V., Ji F., Mishra S., Ma A., Kaniskan H.Ü., Jin J., Lawrence M.S., Sadreyev R.I, & Whetstine J.R. (2019). Histone Lysine Methylation Dynamics Control EGFR DNA Copy Number Amplification. Cancer discovery, 10(2), 306-325.
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9

PBMC Profiling and Single-Cell Sequencing

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For profiling of healthy cells, PBMCs were isolated from fresh blood within 2 hours of collection, using Ficoll-Paque density gradient centrifugation as previously described (58 (link)). Single-cell suspensions were stained per manufacturer recommendations with different panels of antibodies (table S12) designed to enrich for certain population for single-cell sorting and single-cell RNA-sequencing (scRNA-seq) (6 ). Flow cytometry and FACS-sorting of PBMC was performed on a BD Fortessa or BD FACS Fusion instrument, and data analyzed using FlowJo v10.1. Single-cells were sorted into 96-well full-skirted Eppendorf plates chilled to 4°C, pre-prepared with lysis buffer consisting of 10μl of TCL buffer (Qiagen) supplemented with 1% β-mercaptoethanol. Single-cell lysates were sealed, vortexed, spun down at 300 g at 4°C for 1 min, immediately placed on dry ice and transferred for storage at −80°C. Tonsil was mechanically disrupted to obtain single-cell suspension.
Villani A.C., Satija R., Reynolds G., Sarkizova S., Shekhar K., Fletcher J., Griesbeck M., Butler A., Zheng S., Lazo S., Jardine L., Dixon D., Stephenson E., Nilsson E., Grundberg I., McDonald D., Filby A., Li W., De Jager P.L., Rozenblatt-Rosen O., Lane A.A., Haniffa M., Regev A, & Hacohen N. (2017). Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes and progenitors. Science (New York, N.Y.), 356(6335), eaah4573.
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10

Isolation and Sequencing of MAIT Cells

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Intact biopsies from HC and CD patients (with paired inflamed and non-inflamed colon specimens from the latter only) were thawed and enzymatically digested in RPMI supplemented with HEPES, PSG, BCS, MgCl2, CaCl2, Collagenase Type 1, and 0.011% DNase for 30 minutes at 37° C. Cells were then pelleted, resuspended, and mechanically digested by passing the suspension through an 8-gauge needle and filtered through a 100-μum screen. Cells were again pelleted, stained as above, and immediately sorted using the BD FACS Fusion. Live single MAIT cells were sorted into a 96-well plate and their TCR alpha and beta chain amplified using a nested-PCR protocol [70 (link)]. PCR products were Sanger sequenced by a commercial third party (GeneWiz; now Azenta Life Sciences, Chelmsford, MA) using primers specific to the common region of the TCR alpha or beta gene, and TCR alignment was performed using the IMGT HighV-quest tool. For blood TCR analyses, up to 50,000 MAIT cells were sorted by FACS as above from thawed PBMC. DNA extracted from sorted MAIT cells was then sent for comprehensive TCR beta chain sequencing by a commercial third party (Adaptive Biotechnologies, Seattle, WA), with data thus generated retrieved from and analyzed on the latter’s proprietary online ImmunoSEQ Analyser software.
Konecny A.J., Shows D.M, & Lord J.D. (2023). Colonic mucosal associated invariant T cells in Crohn’s disease have a diverse and non-public T cell receptor beta chain repertoire. PLOS ONE, 18(11), e0285918.
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