Lsrii analyser

Manufactured by BD

Sourced in United States

The LSRII analyser is a flow cytometry instrument manufactured by BD. It is designed to detect and analyze the physical and fluorescent characteristics of cells or other particles in a fluid sample. The LSRII provides high-speed data acquisition and analysis capabilities, allowing users to perform a variety of cell-based assays and measurements.

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Lab products found in correlation

Cd8 clone 53 6, by BioLegend (6 mentions) Thy1.1 clone ox 7, by BioLegend (6 mentions) Lsr 2 flow cytometer, by BD (3 mentions) Clone n418, by BioLegend (3 mentions) Thy1.2 clone 30 h12, by BioLegend (3 mentions) Cd4 clone gk1, by BioLegend (3 mentions) Rbc lysis buffer, by Thermo Fisher Scientific (3 mentions) Heparinized glass capillaries, by Thermo Fisher Scientific (3 mentions) Lipofectamine 3000 transfection kit, by Thermo Fisher Scientific (3 mentions) Celltrace violet proliferation kit, by Thermo Fisher Scientific (3 mentions) Cd44 clone im7, by BioLegend (3 mentions) Vα2 clone b20, by BD (3 mentions) Legendplex human th cytokine panel 13 plex, by BioLegend (1 mentions) Soluble anti cd28 antibody, by BD (1 mentions) Cd4 alexa fluor 700 rpa t4, by BD (1 mentions) Cd3 pacific blue ucht1, by BD (1 mentions) Il 32γ, by R&D Systems (1 mentions) Anti cd45, by BioLegend (1 mentions) Anti foxp3, by BioLegend (1 mentions) Anti cd44, by BD (1 mentions)

Spelling variants of this product

LSRII FACS analyser (4 citations) LSRII Fortessa analyser (4 citations) BD LSRII Fortessa cell analyser (2 citations)

33 protocols using lsrii analyser

Mitochondrial Membrane Potential Assay

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Twenty‐four hours after transfection and preceding CCCP treatment, cells were loaded with Rhodamine 123 (0.05 mg/ml, 15 min). Following treatment, the cells were collected for fluorescence‐activated cell sorting (FACS) on the BD LSR II Analyser, with excitation and emission settings of 488 and 530 nm, respectively. Thirty thousand events were recorded per sample, and the geometric mean of the fluorescence intensity values was analysed.

Cummins N., Tweedie A., Zuryn S., Bertran‐Gonzalez J, & Götz J. (2018). Disease‐associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria. The EMBO Journal, 38(3), e99360.

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Isolation and Characterization of Adipose-Derived Stem Cells

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Precursor isolation is described in [18] . Briefly, stromal-vascular fraction (SVF) was prepared from each fat pad by collagenase treatment. Cells were then pelleted by centrifugation, cleared, and suspended in staining media (HBSS + 2% FBS), and labeled with appropriate antibodies (Supplementary Table 2). After staining, cells were filtered through a 35-μm cell-strainer capped tube to ensure single cell suspension and stained with live/dead Blue. Live single cells were gated according to the expression of surface markers (CD31-CD45-CD29 + CD34 + Sca1+) in a BD LSRII analyser. Data were analyzed with FlowJo.

Sanchez-Gurmaches J., Martinez Calejman C., Jung S.M., Li H, & Guertin D.A. (2019). Brown fat organogenesis and maintenance requires AKT1 and AKT2. Molecular Metabolism, 23, 60-74.

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Cytokine Analysis of CD4+ T-cell Activation

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CD4⁺ T-cells were isolated from total PBMCs by negative selection using EasySep Human CD4 T Enrichment columns (StemCell, Cat #19052). Purity of CD4 was typically >98% as determined by surface staining of CD4⁺ T-cells with CD3-Pacific Blue (UCHT1) and CD4-Alexa Fluor 700 (RPA-T4), (both from BD) and FACS analysis (BD LSRII analyser). Purified CD4⁺ T-cells were stimulated with plate-coated anti-CD3 antibody (0.5μg/ml) and soluble anti-CD28 antibody (0.5μg/ml) (both from BD Biosciences, Cat ##555329, 5555726 respectively) for 48 h in the presence or absence of 500 ng IL-32α (BioLegend, Cat # 551004) or IL-32γ (R&D Cat # 4690-IL-025/CF). Secreted cytokines were measured from the supernatant of activated cells using the LEGENDplex™ Human Th Cytokine Panel (13-plex) (BioLegend, Cat # 740001) according to manufacturer’s directions. Infections of PBMCs stimulated with PHA and IL-2 (0.25μg/ml, and 100 units/ml, respectively) or resting cells were carried out using 50 ng of the laboratory strain HIV-BaL per 10⁶ cells by Spinoculation61 (link) (2 hours/Room temperature). Following infection, PBMCs were washed twice by PBS and centrifuged at 1500 rpm for 5 min then were re-suspended in RPMI-1640 medium supplemented with 10% FBS. Cells were kept in culture for 48 h before collection of supernatants and cells for cytokine measures.

El-Far M., Kouassi P., Sylla M., Zhang Y., Fouda A., Fabre T., Goulet J.P., van Grevenynghe J., Lee T., Singer J., Harris M., Baril J.G., Trottier B., Ancuta P., Routy J.P., Bernard N, & Tremblay C.L. (2016). Proinflammatory isoforms of IL-32 as novel and robust biomarkers for control failure in HIV-infected slow progressors. Scientific Reports, 6, 22902.

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Endothelial Cells Transduction by RBC-AAV

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EA.hy926 human endothelial cells were seeded in a 6‐well plate at 200 000 cells/well, and incubated overnight prior to treatment. Cells were treated for 14 days with an equivalent dose of either free AAV‐eGFP or AAV‐eGFP recovered from RBC‐AAV‐eGFP by lysing RBCs at different AAV to endothelial ratios. eGFP expression in transduced cells was both visualized with fluorescence microscopy and quantified with flow cytometer (BD LSR II Analyser).

Zhao Z., Kim J., Suja V.C., Kapate N., Gao Y., Guo J., Muzykantov V.R, & Mitragotri S. (2022). Red Blood Cell Anchoring Enables Targeted Transduction and Re‐Administration of AAV‐Mediated Gene Therapy. Advanced Science, 9(24), 2201293.

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Multiparametric Flow Cytometry of Immune Cells

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Different panels of antibody cocktails were generated from anti‐CD45 (BioLegend, 103116, 30‐F11), anti‐CD3 (BioLegend, 100218, 17A2), anti‐CD4 (BioLegend, 100421, GK1x.5), anti‐CD8a (BioLegend, 100711, 53‐6.7), anti‐NKp46 (BioLegend, 137606, 29A1.4), anti‐CD11c (BioLegend, 117307, N418), anti‐CD25 (Biolegend, 101908, 3C7), anti‐FOXP3 (Biolegend, 126404, MF‐14), anti‐granzyme B (BioLegend, 372208, QA16A02), anti‐IFNγ (BioLegend, 505849, XMG1.2), anti‐IFNγ (BioLegend, 505806, XMG1.2, anti‐CD62L (BioLegend, 104432, MEL‐14) and anti‐CD44 (BD Biosciences, 560568, IM7) antibodies, and the AmCyan Live/Dead Cell Staining Kit (Thermo Fisher Scientific). All antibodies were diluted at optimized dilutions before being used.
Female 7–8 weeks old C57BL/6 mice (n = 4–5 per group) were intravenously administered with the equivalent dose (3.8 × 10¹⁰ vg/mouse) of either free AAV‐fLuc or RBC‐AAV‐fLuc. Mice were then euthanized on day 31, and spleen and lungs were collected. Organ dissociation kits (Miltenyi Biotec) were used per manufacturer's instruction to generate a single‐cell suspension from excised organs, and cells were stained with the antibodies listed above and analyzed using flow cytometry (BD LSR II Analyser). Flow cytometry data analyses were performed using FlowJo 10 software.

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Tracking T Cell Responses to BMDC Transplant

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Thy1.1⁺/1.2⁺ P14 T cells and Thy1.1⁺/1.1⁺ SMARTA T cells were isolated from TCR Tg mice as detailed above. 10⁵ P14 T cells and 10⁵ SMARTA T cells were co-transferred I.V. by retro-orbital injection into wild-type B6 recipients 0–19 days prior to BMDC transplant.
BMDCs were prepared from the bone marrow of NINJA mice as described in^{47 (link)}. Four days after bone marrow harvest, maturing BMDCs were transduced in vitro with 10¹⁰ PFU/mL of recombinant Ad5CMVFLPo (Iowa Vector Core, VVC-U of Iowa-530HT) or 3.5 × 10⁸ PFU/mL of recombinant Ad5CMVeGFP (Iowa Vector Core, VVC-U of Iowa-4). BMDCs were harvested 3 days later and stained with antibodies specific for CD11c (clone N418, BioLegend) and MHC-I (H-2Kb, clone AF6–88.5.5.3, eBioscience) and analysed by flow cytometry on a BD LSRII analyser (BD Biosciences). 10⁴ CD11c⁺MHC-I⁺GFP⁺ BMDCs were then transplanted into the footpad of B6 hosts in 15 μls PBS.
Recipient animals were euthanized 7 days after BMDC transplant to collect spleen and the draining LN (popliteal). Organs were processed as described in^{5 (link)} and samples were stained with antibodies specific for Thy1.1 (clone OX-7, BioLegend), Thy1.2 (clone 30-H12, BioLegend), CD4 (clone GK1.5, BioLegend) and CD8 (clone 53–6.7, BioLegend). Cells were then analysed on a BD LSRII flow cytometer (BD Biosciences).

Damo M., Fitzgerald B., Lu Y., Nader M., William I., Cheung J.F., Connolly K.A., Foster G.G., Akama-Garren E., Lee D.Y., Chang G.P., Gocheva V., Schmidt L.M., Boileve A., Wilson J.H., Cui C., Monroy I., Gokare P., Cabeceiras P., Jacks T, & Joshi N.S. (2020). Inducible de novo expression of neoantigens in tumor cells and mice with NINJA. Nature biotechnology, 39(1), 64-73.

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Isolation and Characterization of Adipose Progenitor Cells

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Adipose progenitor cells (APC) were isolated as follows^{8 (link), 29 (link)}: Stromal-vascular fraction (SVF) was prepared from each fat pad by collagenase treatment and SVF pellets were resuspended in erythrocyte lyses buffer (0.15 M ClNH₄, 0.01 M HCO₃K in water). Cells were then pelleted by centrifugation, resuspended in staining media (HBSS + 2% FBS), and labeled with appropriate antibodies. After staining, cells were filtered through a 35-μm cell-strainer capped tube to ensure single cell suspension and stained with live/dead Blue. Live single cells were gated according to the expression of surface markers (CD31⁻CD45⁻CD29⁺CD34⁺Sca1⁺) in a BD LSRII analyser. Data was analyzed with FlowJo.

Sanchez-Gurmaches J, & Guertin D.A. (2014). Adipocytes arise from multiple lineages that are heterogeneously and dynamically distributed. Nature communications, 5, 4099.

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HR assay in U2OS-DR-GFP cells

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The HR assay was performed in U2OS-DR-GFP cell lines [37 (link),38 (link)]. Cells cultured in 6 well plates at 40% confluency were transfected twice with control or target siRNA. 24 hours after the second round of siRNA transfection, cells were transfected with I-SceI-expressing pCBASce plasmid. After an additional 48 hours of culture, cells were harvested and GFP positive cells were analyzed by flow cytometry (BD LSRII analyser).

Ge C., Che L, & Du C. (2015). The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex. PLoS ONE, 10(12), e0144957.

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Isolation and Characterization of Adipose Progenitor Cells

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Sanchez-Gurmaches J, & Guertin D.A. (2014). Adipocytes arise from multiple lineages that are heterogeneously and dynamically distributed. Nature communications, 5, 4099.

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Multiparametric Flow Cytometry Analysis of Immune Cells

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Single-cell suspensions were treated with anti-CD16/32 (Fc block) (14-9161-73, ThermoFisher) and stained with the live/dead marker ethidium monoazide bromide (ThermoFisher #E1374) in 2% FBS in PBS. The following antibodies were used at a concentration of 1 μg/mL except where otherwise indicated: APC-Cy7-CD117 (clone 2B8; Biolegend #105826), PE-FceRI (clone MAR-1; eBioscience 12-5898-82), eFluor450-CD45 (clone 30-F11; eBioscience #48-0451-82), APC-CD11b (clone M1/70; ThermoFisher #17-0112-82), Alexa700-CD19 (clone 6D5; Biolegend #B189284), PE/Cy7-CD3e (clone 145-SC11, eBioscience #25-0031-82), APC-MHC II (clone M5/114.15.2, eBioscience #17-5321-82), and PE-Siglec F (clone E50-2440, BD Pharmingen #5521126). Cells were fixed with 1.6% paraformaldehyde (Electron Microscopy Sciences, #15710). Flow cytometry was performed using a BD LSRII analyser equipped with the following lasers: 355 nm (UV), 405 nm (violet), 488 nm (blue), and 633 nm (red). Data was analysed using FlowJo software. Gates were drawn according to fluorescence minus one (FMO) controls.

Florsheim E.B., Bachtel N.D., Cullen J., Lima B.G., Godazgar M., Zhang C., Carvalho F., Gautier G., Launay P., Wang A., Dietrich M.O, & Medzhitov R. (2023). Immune sensing of food allergens promotes aversive behaviour. bioRxiv.

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