Lsrii facs analyzer

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The BD LSRII FACS analyzer is a flow cytometry instrument designed for multiparameter analysis of cells and particles. It features multiple laser configurations and detectors for simultaneous measurement of various cellular parameters, including size, granularity, and fluorescence intensity.

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32 protocols using lsrii facs analyzer

Multiparameter Flow Cytometry of B cell Subsets

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Analysis of B cell surface proteins was determined by flow cytometry using PE-594 anti-CD19 (HIB19), Brilliant Violet (BV)605 anti-IgD (IA6–2), BV650-anti-CD27 (O323), PerCP.Cy5 anti-CD38 (HIT2), APC anti-CD21 (Bu32), Pacific Blue anti-CD11c (3.9) obtained from BioLegend, and PE anti-IL-4R (hIL4R-M57) obtained from BD Biosciences. Dead cells were excluded from analysis with APC-eFluor® 780 Organic Viability Dye (ThermoFisher Scientific).
For intracellular staining of IFN-β (clone MMHB-3, PBL Assay Science), cells were stained with ef780 viability dye, followed by fixation in 2% PFA and 70% ice-cold methanol permeabilization before staining. For intranuclear staining of T-bet (4B10, BioLegend) or IRF7 (12G9A36, BioLegend), permeabilization and fixation were carried out using the eBioscience™ Transcription Factor Staining Buffer (ThermoFisher Scientific). Cells (300,000–1 X 10⁶ per sample) were analyzed by flow cytometry. FACS data were acquired with an LSRII FACS analyzer (S/N# 30201809, BD Biosciences) and analyzed with FlowJo software v10.6.2 (Tree Star Ashland, OR).

Gao M., Liu S., Chatham W.W., Mountz J.D, & Hsu H.C. (2022). IL-4-induced quiescence of resting naïve B cells is disrupted in systemic lupus erythematosus. Journal of immunology (Baltimore, Md. : 1950), 209(8), 1513-1522.

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Humanized Mice for Immune Research

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Humanized mice (Hu-mice) were prepared as described in prior publications (61 (link)–63 (link)). Shortly after birth, NOD.Cg-Prkdc^scid Il2rgt^m1Wjl/SzJ mice were briefly irradiated with a sub-lethal dose of radiation (1Gy) using a RS-2000 X-Ray Irradiator (Rad Source Technologies). CD34+ cells (50,000 cells/mouse) enriched from human cord blood (>90%) were then injected intra-hepatically and left for humanization. At monthly intervals mice were bled via a submandibular vein into EDTA-coated tubes and screened for human immune cells using flow cytometry (LSR-II FACS analyzer, BD Biosciences, Mountain View, CA, USA). The CD45 percentage in humanized mice used in this study ranged from 25 to 50%. About 5% of our Hu mice develop graft vs.-host disease (GVHD) and these mice were excluded from our study.

Dash P.K., Alomar F.A., Cox J.L., McMillan J., Hackfort B.T., Makarov E., Morsey B., Fox H.S., Gendelman H.E., Gorantla S, & Bidasee K.R. (2021). A Link Between Methylglyoxal and Heart Failure During HIV-1 Infection. Frontiers in Cardiovascular Medicine, 8, 792180.

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Monocyte NF-κB Phosphorylation and ROS Assay

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The monocytes were pretreated with normal medium or AAF medium overnight, and then stimulated with 10 μg/ml SAA for 15 min. Phosphorylation of NF-κB 65 was analyzed by flow cytometry as described previously (15 (link)). In short, the monocytes were fixed and permeabilized.
We then stained the monocytes with phycoerythrin (PE)-labelled phospho-p65 antibody (BD Biosciences). LSRII FACS analyzer (BD Biosciences) was used to analyze the stained monocytes. To measure ROS levels, treated monocytes were cultured with CM-H2DCFDA (Invitrogen, Carlsbad, CA, USA) for 30 min at 37°C, and tested by flow cytometry.

Yu N., Peng C., Chen W., Sun Z., Zheng J., Zhang S., Ding Y, & Shi Y. (2021). Circulating Metabolomic Signature in Generalized Pustular Psoriasis Blunts Monocyte Hyperinflammation by Triggering Amino Acid Response. Frontiers in Immunology, 12, 739514.

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Multiparameter Flow Cytometry Profiling

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Fluorochrome conjugated antibodies were purchased from Biolegend (anti-CD45 Cat# 103132, anti-CD4 Cat# 100546, anti-CD8 Cat# 100714, anti-NK1.1 Cat# 108710). Aqua fluorescent reactive dye (live/dead) was purchased from Invitrogen (Cat# L34966A), and Fc block was purchased from Biolegend (Cat# 101320) was performed. Flow cytometric analysis was performed on LSR II FACS analyzer (BD Biosciences) at the Saint Louis University Flow Cytometry Core Facility. Live lymphocytes were identified as staining negative for live/dead stain and positive for CD45. These were segregated into CD4⁺ and CD8⁺ T cells or CD4/8-negative NK1.1⁺ natural killer (NK) cells. CD4⁺ T cells were further segregated into conventional FoxP3-negative and Foxp3⁺ regulatory cells. Flow cytometry data was analyzed using FlowJo v.10 software (Tree Star Inc.).

Kuehm L.M., Khojandi N., Piening A., Klevorn L.E., Geraud S.C., McLaughlin N.R., Griffett K., Burris T.P., Pyles K.D., Nelson A.M., Preuss M.L., Bockerstett K.A., Donlin M.J., McCommis K.S., DiPaolo R.J, & Teague R.M. (2020). Fructose promotes cytoprotection in melanoma tumors and resistance to immunotherapy. Cancer immunology research, 9(2), 227-238.

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Isolation and Immunostaining of Mouse Spinal Cord Nuclei

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Nuclei of mouse spinal cords were isolated as described above. After filtering of the homogenate, nuclei were fixed with methanol for 10 min on ice, followed by two washing steps with nuclei incubation buffer. For intranuclear staining, nuclei were permeabilized with nuclei incubation buffer supplemented with 0.5% Triton X-100 for 15 min on ice. After blocking with nucleus incubation buffer and 10% NDS, primary antibody was added for 1 hour at room temperature, followed by a washing step and subsequent incubation in secondary antibody for 30 min at room temperature. Immunofluorescence was acquired on an LSR II FACS analyzer (BD Biosciences). Data analysis was performed with the FlowJo v.10 analysis software (FlowJo LLC).

Rothammer N., Woo M.S., Bauer S., Binkle-Ladisch L., Di Liberto G., Egervari K., Wagner I., Haferkamp U., Pless O., Merkler D., Engler J.B, & Friese M.A. (2022). G9a dictates neuronal vulnerability to inflammatory stress via transcriptional control of ferroptosis. Science Advances, 8(31), eabm5500.

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Comprehensive Multicolor Flow Cytometry

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Cells were stained for surface markers with the following antibodies: Pacific-blue- or Alexa-488-anti-CD4 (RM4-5, GK1.5); Pacific-blue-anti-CD19 (6D5); PE conjugated anti-PD-1 (RMP1-30), CD44 (IM7), TGF-β1 (TW7-16B4) all from Biolegend. Alexa-647-anti-GL-7 (GL7); FITC- or PE-anti-ICOS (398.4A or 7E.17G9); PE conjugated anti-CD25 (pc61.5), GITR (DTA-1), and Fas (15A7) all from eBioscience; PE-Cy7-anti-CXCR5 (2G8, BD Biosciences); PE-anti-CTLA-4 (UC10-4F10-11, BD Pharmingen).
For nuclear transcription factor staining, cells were labeled with surface markers, then fixed and permeabilized with the Foxp3-Staining-Buffer-Set (eBioscience), according to the manufacturer's instruction. Cells were then stained with PE-anti-Bcl6 (K112-91, BD Biosciences) and PE-anti-Foxp3 (FJK-16s, eBiosciences).
For phospho-flow staining, after treatment, cells were fixed and permeabilized with the BD Phosflow™ Fix Buffer and Perm Buffer, according to the manufacturer's instruction. Surface markers staining were followed by intracellular staining with Alexa-647-rabbit-anti-phospho-Akt-Ser473 (Cell signaling) or Pacific-blue-mouse-anti-Stat3-p-Y705 (4/p-Stat3, BD Bioscience). Samples were acquired with an LSRII FACS analyzer (BD Biosciences), and data was analyzed with FlowJo software (Tree Star, Inc. Ashland, OR, USA).

Ding Y., Li J., Yang P., Luo B., Wu Q., Zajac A.J., Wildner O., Hsu H.C, & Mountz J.D. (2014). Interleukin-21 Promotes Germinal Center Reaction by Skewing the Follicular Regulatory T Cell to Follicular Helper T Cell Balance in Autoimmune BXD2 Mice. Arthritis & rheumatology (Hoboken, N.J.), 66(9), 2601-2612.

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Quantifying Antigen-Specific T Cell Recall

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For antigen-specific recall assays, 9 d after immunization of the mice, 2.5 × 10⁵ draining inguinal lymph node cells were prepared and cultured in 96-well round-bottom plates for 72 h with the indicated concentrations of MOG_35–55 peptide, a vehicle control, or plate-coated anti-CD3ε (1 µg/ml; BioLegend; catalog no. 100340) together with soluble anti-CD28 (1 µg/ml; BioLegend; catalog no. 102116) as a positive control. During the last 16 h of culture, cells were pulsed with 1 µg/ml BrdU (catalog no. 423401). Single-cell suspensions were stained for surface antigens in the presence of TruStain Fc receptor block (BioLegend), and Fixable Viability Stain 780 (BD Biosciences; catalog no. 565388) was used to discriminate dead cells. Cells were fixed (fixation buffer; BioLegend; catalog no. 420801) and permeabilized using 0.5% Triton X-100, followed by incubation with 40 KU/ml DNase I (Merck; catalog no. 260913-10MU) in PBS with Ca²⁺ and Mg²⁺ for 1 h at 37°C. After DNA digestion, incorporated BrdU was detected by incubation with an anti-BrdU AF647-coupled antibody. The antibodies and the respective antigen, host species, supplier, catalog number, clone, and dilution are listed in Table S8. Data were acquired on an LSR II FACS analyzer (BD Biosciences). Representative gating strategies will be provided upon request.

Woo M.S., Ufer F., Rothammer N., Di Liberto G., Binkle L., Haferkamp U., Sonner J.K., Engler J.B., Hornig S., Bauer S., Wagner I., Egervari K., Raber J., Duvoisin R.M., Pless O., Merkler D, & Friese M.A. (2021). Neuronal metabotropic glutamate receptor 8 protects against neurodegeneration in CNS inflammation. The Journal of Experimental Medicine, 218(5), e20201290.

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Analyzing Cell Surface Markers

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NTC and sh1/sh2 cells were fixed, permeabilized, and probed for primary-tagged anti-ITGB1 and anti-CD44 as discussed (23 ). The samples were analyzed using a LSR II FACS analyzer (BD Biosciences).

Ray U., Jung D.B., Jin L., Xiao Y., Dasari S., Sarkar Bhattacharya S., Thirusangu P., Staub J.K., Roy D., Roy B., Weroha S.J., Hou X., Purcell J.W., Bakkum-Gamez J.N., Kaufmann S.H., Kannan N., Mitra A.K, & Shridhar V. (2022). Targeting LRRC15 Inhibits Metastatic Dissemination of Ovarian Cancer. Cancer Research, 82(6), 1038-1054.

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Multiparameter Flow Cytometry Profiling

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Human antibodies included BioLegend BV510-anti-CD24 (ML5), PE-anti-CD303 (clone 201A), BV510-anti-IgM (clone MHM-88), Pacific-Blue-anti-CD4 (clone RPA-T4), PE-Cy7-anti-CD10 (clone HI10a), BV650-anti-CD27 (O323), Pacific Blue-anti-CD19 (HIB19), PE-Cy7-anti-CD38 (HB-7); Southern Biotech PE-IgD (IADB6), and PBL Assay Science FITC-anti-IFNβ (MMHB-3). Dead cells were excluded from analysis with APC-eFluor® 780 Organic Viability Dye (eBioscience). For intracellular staining, cells were stained with ef780 viability dye, followed by fixation in 2% PFA and 70% ice-cold methanol permeabilization prior to staining. Purity was validated by post-sort analysis of FACS sorted cells to verify that >99% of cells fell into the sort gate after resorting. FACS data were acquired with an LSRII FACS analyzer (BD Biosciences) and analyzed with FlowJo software (Tree Star Ashland, OR).

Hamilton J.A., Wu Q., Yang P., Luo B., Liu S., Li J., Mattheyses A., Sanz I., Chatham W.W., Hsu H.C, & Mountz J.D. (2018). Intracellular interferon-β and distinct type I IFN expression patterns in circulating SLE B cells. Journal of immunology (Baltimore, Md. : 1950), 201(8), 2203-2208.

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Transduction of Postnatal Thymocytes

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For transduction experiments, CD34⁺CD1a⁻ postnatal thymocytes were cultured overnight in Yssel’s medium (29 (link)) with 5% normal human serum, 20 ng/ml SCF and 10 ng/ml IL-7. The following day cells were incubated for 6–7 h with virus supernatant in retronectin-coated plates (30 µg/ml; Takara Biomedicals, Shiga, Japan). The development of ILCs and NK cells was assessed by coculturing the mixture of transduced and non-transduced CD34⁺CD1a⁻ progenitor cells with OP9 cells in MEMα medium (Life Technologies, Carlsbad, CA, USA) with 20% FCS (Hyclone Laboratories, Logan, UT, USA), 5 ng/ml SCF, 5 ng/ml IL-7, and 5 ng/ml FLT3L. 0.5 ng/ml IL-15 was added at the onset of the culture, the medium containing IL-15 was refreshed every week. Flow cytometric analyses were performed on a LSRII FACS analyzer (BD Biosciences); electronic gating was performed using FlowJo (Tree Star, Ashland, OR, USA). Numbers in each dot plot represent the percentage of cells in each quadrant. The fold expansion in absolute cell numbers was calculated using Microsoft Excel 2007 (Microsoft, Redmond, WA, USA) on the basis of total numbers of cells harvested from the cultures, percentages of transduced cells, and percentages of each population corrected for the number of input cells.

Nagasawa M., Germar K., Blom B, & Spits H. (2017). Human CD5+ Innate Lymphoid Cells Are Functionally Immature and Their Development from CD34+ Progenitor Cells Is Regulated by Id2. Frontiers in Immunology, 8, 1047.

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