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Lsrii analytical flow cytometer

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The BD-LSRII is an analytical flow cytometer designed for high-performance multiparameter analysis. It is capable of detecting and analyzing a wide range of cellular parameters, including size, granularity, and the expression of multiple fluorescently labeled markers on single cells in a complex sample.

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Lab products found in correlation

Apc h7 2d1, by BD (2 mentions) Pecy7 581, by BD (2 mentions) Apc hb7, by BD (2 mentions) Af 405 8g10nb, by Novus Biologicals (2 mentions) Mouse igg2b mpc11, by Novus Biologicals (2 mentions) 7 amino actinomycin, by Thermo Fisher Scientific (2 mentions) Mr frosty freezing container, by Thermo Fisher Scientific (1 mentions) Ack lysing buffer, by Lonza (1 mentions) Alexa fluor 700, by BD (1 mentions) Facsdiva software, by BD (1 mentions) Fcs express version 3, by De Novo Software (1 mentions) Alexa fluor 546, by Thermo Fisher Scientific (1 mentions) Facsdiva acquisition software, by BD (1 mentions) Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions) Fcs express 3, by De Novo Software (1 mentions) Click it plus edu alexa fluor 647 flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions) Multicycle av, by Phoenix Pharmaceuticals (1 mentions) Countbright beads, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

6-laser LSRII analytical flow cytometer BD LSRII analytical cytometer LSRII flow cytometer LSRII cytometer

13 protocols using lsrii analytical flow cytometer

1

Isolation and Characterization of Lymph Node Mononuclear Cells

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Inguinal lymph nodes were teased apart and used to isolate LNMC (lymph node mononuclear cells). LNMC were washed with ice-cold PBS containing 10% heat-inactivated FBS, and centrifuged at 300 × g for 10 min at 4°C. ACK lysing buffer (Lonza) was added to pelleted cells to remove erythrocytes. Isolated LNMC were placed in heat-inactivated FBS with 10% DMSO, slow-frozen in a Mr. Frosty™ freezing container (Thermo Scientific) at −80°C for 24 hours, and stored in a liquid nitrogen dewar.
For determination of SMX-adducts by flow cytometry, LNMC were thawed at 37°C and washed with PBS containing 10% FBS; 500,000 cells were stained with polyclonal rabbit anti-SMX sera (1:30,000), along with polyclonal goat anti-rabbit IgG linked with FITC (1:20; BD Biosciences). Validation experiments for detecting B cells revealed excessive background without the primary antibody, so subsequent experiments were focused on T cell populations. T cells were identified with specific antibodies for macaque CD3 (linked to Alexa Fluor 700; BD Biosciences) clone SP34-2, CD4 (linked to APC; Miltenyi Biotec Inc) clone M-T466, and CD8 (linked to Pacific Blue; BD Biosciences) clone RPA-T8. Reactions were analyzed for surface SMX adducts in lymphocyte subpopulations on an LSR II analytical flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (TreeStar, Ashland, OR).
Wong Y.Y., Rakasz E.G., Gasper D.J., Friedrich T.C, & Trepanier L.A. (2016). Immunogenicity of trimethoprim/sulfamethoxazole in a macaque model of HIV infection. Toxicology, 368-369, 10-18.
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2

Immunophenotyping of Macrophages and Cancer Cells

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Human M-CSF-induced macrophages and DLD-1 cells were washed then lifted with 10 mM EDTA (pH 8). Cells were incubated with LIVE/DEAD™ Fixable Violet viability dye for 15 min on ice followed by fixation with 4% paraformaldehyde (PFA). Cells were permeabilized with permeabilization buffer and incubated with human Fc block (10 µg/ml) for 15 min53 (link). Next, cells were incubated with anti-Robo-1 Ab used at 10 µg/ml for 30 min. Cells were washed then incubated with AF-594-conjugated donkey anti-rabbit Ab used at 1 µg/ml for 15 min. All labelling steps were performed on ice. Samples were acquired using an LSRII analytical flow cytometer (BD Biosciences-US) with FACSDiva software (BD Biosciences-US) and analyzed with FlowJo v10 (BD Biosciences-US).
Yusuf B., Mukovozov I., Patel S., Huang Y.W., Liu G.Y., Reddy E.C., Skrtic M., Glogauer M, & Robinson L.A. (2021). The neurorepellent, Slit2, prevents macrophage lipid loading by inhibiting CD36-dependent binding and internalization of oxidized low-density lipoprotein. Scientific Reports, 11, 3614.
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3

Erythroblast Iron Uptake Mechanisms

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Erythroblasts (5 × 105) were incubated with 5 μg human diferric Tf conjugated to Alexa Fluor™ 546 (Thermo Fisher Scientific, Cat. No. T23364) or 3 μg of recombinant H-Ft conjugated to R-phycoerythrin (H-Ft-PE; Creative Biomart, Shirley, NY, USA, Cat. No. FTH1-528H-PE) for 60 min on ice. Competing antibodies were added to some samples and included 10 μg of either anti-dansyl IgG1/κ isotype control antibody67 (link), ch128.1/IgG1, or the murine anti-hTfR1 IgG2a/κ antibody clone M-A712 (BD Biosciences, Cat. No. 555534) that is known to block H-Ft binding33 (link). Erythroblasts incubated with buffer (PBS) served as a negative unstained control. After the incubation, cells were washed, fixed in 1% paraformaldehyde in PBS and analyzed by flow cytometry using an LSRII analytical flow cytometer and the FACSDiva™ acquisition software (version 8.0.3; BD Biosciences, San Diego, CA, USA). Histograms were created using FCS Express version 3.0 (De Novo Software, Pasadena, CA, USA).
Hickerson B.T., Daniels-Wells T.R., Payes C., Clark L.E., Candelaria P.V., Bailey K.W., Sefing E.J., Zink S., Ziegenbein J., Abraham J., Helguera G., Penichet M.L, & Gowen B.B. (2022). Host receptor-targeted therapeutic approach to counter pathogenic New World mammarenavirus infections. Nature Communications, 13, 558.
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4

Mitochondrial Membrane Potential Assay

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Cells were seeded at a density of 85,000 cells/well of a six‐well plate and treated with 500 nM Tg for 3 h prior to collection. CCCP (10 μM) was added 50 min before collection, followed by 200 nM TMRE (Thermofisher) 20 min before collection. Samples were collected using TrypLE Express and cell culture media. Following a brief centrifugation, cell pellets were washed in DPBS (Gibco) and resuspended in DPBS supplemented with 5% BSA. Fluorescence intensity of TMRE for 20,000 cells/condition was recorded on the PE channel of a BD Biosciences LSR II analytical flow cytometer. Data are presented as geometric mean of the fluorescence intensity from three experiments normalized to vehicle‐treated cells.
Perea V., Cole C., Lebeau J., Dolina V., Baron K.R., Madhavan A., Kelly J.W., Grotjahn D.A, & Wiseman R.L. (2023). PERK signaling promotes mitochondrial elongation by remodeling membrane phosphatidic acid. The EMBO Journal, 42(15), e113908.
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5

Flow Cytometry Analysis of BMDC Viability

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A previously used protocol from our group was employed with modifications to assess day 6 mature BMDC viability and phenotype [26 (link)] using the fluorescent antibodies and dyes listed in Supplemental Table S1. Zombie NIR™ fixable vital dye was added to BMDC at room temperature for 20 min in PBS to determine viability. Next, BMDC underwent surface immunofluorescence staining in the presence of TruStain FcX™ anti-mouse CD16/CD32 block at 4 °C for 25 min. Extensive washing in HBSS and 0.1% bovine serum albumin ensued prior to fixation in 4% paraformaldehyde and storage at 4 °C. When necessary, eGFP expression due to Ad transduction and fluorescent membrane-intercalating dye incorporation (detailed below) was assessed using flow cytometry and compared to an aliquot of nonfluorescent BMDC. Data was acquired on a LSRII analytical flow cytometer (BD Biosciences, San Jose, USA) and analyzed using FlowJo Software (v10, Tree Star, Inc., Ashland, USA).
Fink C., Gevaert J.J., Barrett J.W., Dikeakos J.D., Foster P.J, & Dekaban G.A. (2023). In vivo tracking of adenoviral-transduced iron oxide-labeled bone marrow-derived dendritic cells using magnetic particle imaging. European Radiology Experimental, 7, 42.
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6

Evaluating Antibody Binding to CD38 and FcεRI

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The specificity of the antibody variable region for human CD38 and binding of the Fc region to human FcεRI were assessed using flow cytometry. 5 × 105 MM.1S cells expressing CD38 or RBL SX-38 expressing human FcεRI were incubated with either vehicle (RPMI + 10% FBS), 2 μg IgE isotype control (non-targeting IgE antibody), anti-CD38 IgG1, or anti-CD38 IgE in 100 μL of RPMI with 10% FBS on ice for 1 h. Cells were washed with RPMI with 10% FBS and incubated for 30 min on ice with a phycoerythrin (PE)-conjugated goat F(ab′)2 anti-human κ antibody (Thermo Fischer Scientific) to detect primary antibody binding. Samples (104 events per sample) were analyzed on a BD LSRII analytical flow cytometer (BD Biosciences, San Jose, CA, USA) and histograms were generated using FCS Express 3 (De Novo Software, Los Angeles, CA, USA).
Candelaria P.V., Nava M., Daniels-Wells T.R, & Penichet M.L. (2023). A Fully Human IgE Specific for CD38 as a Potential Therapy for Multiple Myeloma. Cancers, 15(18), 4533.
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7

Quantifying Human Cell Engraftment

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To assess human cell engraftment in BM was harvested at the indicated time point. Cells were stained with anti-human CD45-APCH7 (hCD45, 2D1), anti-mouse CD45-PECy5 (mCD45; 3OF11, BioLegend, San Diego, CA, USA). Human cells were determined by evaluating the percentage of hCD45+mCD45- cells. Cells were evaluated in a BD-LSRII analytical flow cytometer.
Trujillo-Alonso V., Pratt E.C., Zong H., Lara-Martinez A., Kaittanis C., Rabie M.O., Longo V., Becker M.W., Roboz G.J., Grimm J, & Guzman M.L. (2019). FDA-approved ferumoxytol displays anti-leukaemia efficacy against cells with low ferroportin levels. Nature nanotechnology, 14(6), 616-622.
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8

Quantifying Human Cell Engraftment

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To assess human cell engraftment in BM was harvested at the indicated time point. Cells were stained with anti-human CD45-APCH7 (hCD45, 2D1), anti-mouse CD45-PECy5 (mCD45; 3OF11, BioLegend, San Diego, CA, USA). Human cells were determined by evaluating the percentage of hCD45+mCD45- cells. Cells were evaluated in a BD-LSRII analytical flow cytometer.
Trujillo-Alonso V., Pratt E.C., Zong H., Lara-Martinez A., Kaittanis C., Rabie M.O., Longo V., Becker M.W., Roboz G.J., Grimm J, & Guzman M.L. (2019). FDA-approved ferumoxytol displays anti-leukaemia efficacy against cells with low ferroportin levels. Nature nanotechnology, 14(6), 616-622.
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9

Ferroportin Expression in Primary AML

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For primary AML samples, cells were stained with anti-human CD45 conjugated to allophycocyanin-cyanine (APC-H7; 2D1, BD Biosciences, Franklin Lakes, NJ, USA), CD34 conjugated to phycoerythrin-cyanine-7 (PECy7; 581, BD Biosciences, Franklin Lakes, NJ, USA ) and CD38 conjugated to allophycocyanin (APC; HB7, BD Biosciences, Franklin Lakes, NJ, USA ) and Ferroportin (FPN) conjugated to Alexa Fluor-405 (AF-405; 8G10NB, Novus Biologicals, Littleton, CO, USA) or isotype control AF-405 (mouse IgG2b; MPC11 (Novus Biologicals, Littleton, CO, USA )) for 30 min in the dark at 4oC. For cell lines, cells were only stained with FPN-AF-405 or Isotype control for 30 min in the dark at 4oC. Cells were washed two times and resuspended in cold FACS buffer containing a viability dye (7-aminoactinomycin; 7AAD, Life Technologies, Carlsbad, CA, USA). Cells were evaluated in a BD-LSRII analytical flow cytometer. Analysis was later performed using FlowJo 9.3 software for Mac OS X (TreeStar, Ashland, OR).
Trujillo-Alonso V., Pratt E.C., Zong H., Lara-Martinez A., Kaittanis C., Rabie M.O., Longo V., Becker M.W., Roboz G.J., Grimm J, & Guzman M.L. (2019). FDA-approved ferumoxytol displays anti-leukaemia efficacy against cells with low ferroportin levels. Nature nanotechnology, 14(6), 616-622.
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10

Ferroportin Expression in Primary AML

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For primary AML samples, cells were stained with anti-human CD45 conjugated to allophycocyanin-cyanine (APC-H7; 2D1, BD Biosciences, Franklin Lakes, NJ, USA), CD34 conjugated to phycoerythrin-cyanine-7 (PECy7; 581, BD Biosciences, Franklin Lakes, NJ, USA ) and CD38 conjugated to allophycocyanin (APC; HB7, BD Biosciences, Franklin Lakes, NJ, USA ) and Ferroportin (FPN) conjugated to Alexa Fluor-405 (AF-405; 8G10NB, Novus Biologicals, Littleton, CO, USA) or isotype control AF-405 (mouse IgG2b; MPC11 (Novus Biologicals, Littleton, CO, USA )) for 30 min in the dark at 4oC. For cell lines, cells were only stained with FPN-AF-405 or Isotype control for 30 min in the dark at 4oC. Cells were washed two times and resuspended in cold FACS buffer containing a viability dye (7-aminoactinomycin; 7AAD, Life Technologies, Carlsbad, CA, USA). Cells were evaluated in a BD-LSRII analytical flow cytometer. Analysis was later performed using FlowJo 9.3 software for Mac OS X (TreeStar, Ashland, OR).
Trujillo-Alonso V., Pratt E.C., Zong H., Lara-Martinez A., Kaittanis C., Rabie M.O., Longo V., Becker M.W., Roboz G.J., Grimm J, & Guzman M.L. (2019). FDA-approved ferumoxytol displays anti-leukaemia efficacy against cells with low ferroportin levels. Nature nanotechnology, 14(6), 616-622.
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