Lsr 11 flow cytometer

Manufactured by BD

Sourced in United States

The BD LSR 11 Flow Cytometer is a laboratory instrument designed to analyze and sort cells or particles in a fluid sample. It uses laser technology to detect and measure various characteristics of individual cells or particles as they pass through a detection chamber.

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Lab products found in correlation

Annexin 5 binding buffer, by BioLegend (3 mentions) Annexin 5 apc, by BioLegend (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Annexin 5, by BioLegend (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by BioLegend (2 mentions) Nf κb p65 d14e12, by Cell Signaling Technology (2 mentions) β tubulin af647 9f3, by Cell Signaling Technology (2 mentions) Cd3ε apc 145 2c11, by BD (2 mentions) Nfat1 af488 d43b1, by Cell Signaling Technology (2 mentions) Enzyme free cell dissociation buffer, by Thermo Fisher Scientific (2 mentions) Facsaria cell sorter, by BD (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Accutase cell detachment solution, by Corning (1 mentions) Tox7 kit, by Merck Group (1 mentions) Cell trace violet proliferation dye, by Thermo Fisher Scientific (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions)

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BD LSR flow cytometer (134 citations)

8 protocols using lsr 11 flow cytometer

Flow Cytometry and Cell Sorting of Thymus Tissue

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For flow cytometry analysis of thymus tissue, thymus was dissected and placed in cold PBS. Cells were dissociated through gentle grinding of tissue between frosted slides and strained through a 100μm cell strainer (Falcon 352360). Cells were counted and 1 million were used for staining. Cells were spun at 400×G for 5 minutes and resuspended in Fix/Perm staining buffer (Affymetrix/eBioscience, 00-5523) for 45 minutes. Cells were then spun at 600×G for 5 minutes and washed in 1X Perm buffer then stained in 1X Perm buffer with antibodies for 488 anti-CD3 (BioLegend, 100212), 647 anti-CD4 (Biolegend, 100426), and PE anti-CK8α (BioLegend, 100708). Cells were then washed 2 times in 1X Perm buffer and resuspended in Perm buffer for analysis. Cells were analyzed using a BD LSR 11 flow cytometer and analyzed using FlowJo, with all gates the same between samples.
For cell sorting experiments, cells transfected with GFP plus either a control plasmid, Brd4 or Brd4-SSS492ESE. 5 days after transfection, cells were washed in PBS and incubated for 30 minutes on ice in FACS buffer (10mM HEPES, 5mM EDTA, 2% BSA, in PBS) and then pipetted to dislodge from plate. GFP positive cells were sorted on a BD FACSAria Cell Sorter (BD Biosciences) directly into Trizol (Life Technologies, 15596-026) and RNA was isolated for analysis.

Korb E., Herre M., Zucker-Scharff I., Gresack J., Allis C.D, & Darnell R.B. (2017). Excess translation of epigenetic regulators contributes to Fragile X Syndrome and is alleviated by Brd4 inhibition. Cell, 170(6), 1209-1223.e20.

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Macrophage Viability Assay of Burkholderia cenocepacia

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MDMs were plated at a density of 3x 10⁶/ml in 12-well plates (BD Falcon, 353043). Cysteamine was added 24h before infection. The cells were infected with B. cenocepacia at an MOI of 10. Viability assay was done by FACS analysis using the APC Annexin V apoptosis detection kit with Propidium Iodide (PI) (Biolegend 640932). The macrophages were detached by Accutase cell detachment solution (Corning 25058C1). Cells were collected, washed, and re-suspended in 100 μl of Annexin V Binding Buffer (Biolegend 422201), and then stained with 3 μl of Annexin V and 5 μl PI in the dark for 20 min at room temperature. The percentage of non-viable, apoptotic cells was assessed using flow cytometry (BD LSR 11 Flow Cytometer; BD Bioscience).

Shrestha C.L., Assani K.D., Rinehardt H., Albastroiu F., Zhang S., Shell R., Amer A.O., Schlesinger L.S, & Kopp B.T. (2017). Cysteamine-mediated clearance of antibiotic-resistant pathogens in human cystic fibrosis macrophages. PLoS ONE, 12(10), e0186169.

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Quantifying Apoptosis in MDMs

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MDMs were plated at a density of 1 × 10⁶/ml in 12 well plates. Apoptosis was measured by flow cytometry and fluorescence-activated cell sorting (FACS) analysis using APC Annexin V (Biolegend, 640920), prodidium iodide (BioLegend) and DAPI (Molecular Probes, D1306). MDMs were detached by Accutase solution, collected, washed, and re-suspended in 100 μl of Annexin V Binding Buffer (Biolegend 422201), and then stained with 5 μl of Annexin V and 0.4 µg/ml DAPI for 15 min at room temperature in the dark. As a positive control, untreated CF and non-CF MDMs were exposed to Thapsgargin (0.25 μM) for 12 hours. The percentages of viable and apoptotic cells was assessed using flow cytometry (BD LSR 11 Flow Cytometer; BD Bioscience).

Zhang S., Shrestha C.L, & Kopp B.T. (2018). Cystic fibrosis transmembrane conductance regulator (CFTR) modulators have differential effects on cystic fibrosis macrophage function. Scientific Reports, 8, 17066.

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Evaluating Cellular Cytotoxicity of AR Compounds

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MDMs were plated at a density of 2 × 10 6 /ml in 12 well plates. MDMs were left uninfected or infected with B. cenocepacia at a MOI of 5. AR-13 was added to indicated wells at a concentration of 5 μM. Viability assay was performed by flow cytometry and fluorescence-activated cell sorting analysis using APC Annexin V (Biolegend, 640,920) and DAPI (Molecular Probes, D1306). MDMs were detached by Accutase solution, collected, washed, and re-suspended in 100 μl of Annexin V Binding Buffer (Biolegend 422,201), and then stained with 5 μl of Annexin V and 0.4 μg/ml DAPI for 15 min at room temperature in the dark. The percentages of viable and apoptotic cells was assessed using flow cytometry (BD LSR 11 Flow Cytometer; BD Bioscience). Cytotoxicity of AR compounds on airway epithelial cells was evaluated by measuring lactate dehydrogenase (LDH) release. LDH assay was using TOX-7 kit (Sigma-Aldrich, Saint Louis, MO) following the manufacturer's protocol. Briefly, reactions were measured at 490 nm absorbance and percentage of cell death was compared to the positive control designated 100% cell death obtained with 1% Triton X-100 as previously described [26] .

Assani K., Shrestha C.L., Rinehardt H., Zhang S., Robledo-Avila F., Wellmerling J., Partida-Sanchez S., Cormet-Boyaka E., Reynolds S.D., Schlesinger L.S, & Kopp B.T. (2019). AR-13 reduces antibiotic-resistant bacterial burden in cystic fibrosis phagocytes and improves cystic fibrosis transmembrane conductance regulator function. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society, 18(5).

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Quantifying Activated T-cell Nuclei

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Whole cells were fixed with 4% paraformaldehyde in 1× PBS. Both fixed whole cells and fixed nuclei were permeabilized with 0.3% Triton X-100 in1× PBS and 2% FBS and stained with fluorescent Abs for 15 min at room temperature followed by three washes in 1× PBS and 2% FBS. To sufficiently pellet nuclei, centrifugation was performed at 1000 × g. The following Abs were used for staining: β-tubulin-AF647 (9F3, #3624; Cell Signaling Technology), CD3ε-APC (145–2C11; BD Biosciences), NFAT1-AF488 (D43B1, #14324; Cell Signaling Technology), and NF-κB (p65) (D14E12, #8242; Cell Signaling Technology). Propidium iodide was sourced from Thermo Fisher Scientific. Samples were collected on an LSR 11 flow cytometer (BD Biosciences) and analyzed with FlowJo 10.4.2 (BD Biosciences/Tree Star). After gating on singlets, OT-I nuclei were identified as CellTrace Violet^hi, β-tubulin^lo. At least 10,000 OT-I nuclei events were recorded per sample.

Gallagher M.P., Conley J.M, & Berg L.J. (2018). Peptide Antigen Concentration Modulates Digital NFAT1 Activation in Primary Mouse Naive CD8+ T Cells as Measured by Flow Cytometry of Isolated Cell Nuclei. ImmunoHorizons, 2(7), 208-215.

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Quantifying Activated T-cell Nuclei

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PECAM-1 Expression and Heparin Binding

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REN cell lines were detached from tissue culture plates by enzyme free cell dissociation buffer (Life Technologies), washed with PBS and resupended in PBS with 2% BSA. The cells were incubated with fluorescently tagged anti‐PECAM‐1 antibody on ice for 30 min. Cells were then washed twice with PBS with 2% BSA. FACS was performed on LSR 11 Flow Cytometer (BD Bioscience) and the data analyzed with Flowjo software (Tree Star, Ashland OR). For the assessment of heparin binding, 1 × 10⁶ cells were stained with heparin Cy5.5 (4 μg) for 45 min on ice and washed twice with PBS with 2% BSA and analyzed by flow cytometry. 7‐amino‐actinomycin D (7AAD) was used to identify nonviable cells.

Abraham V., Parambath A., Joe D.S, & DeLisser H.M. (2016). Influence of PECAM‐1 ligand interactions on PECAM‐1‐dependent cell motility and filopodia extension. Physiological Reports, 4(22), e13030.

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Quantifying Tumor Cell Proliferation

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B16-F10 or 4T1 cells (labeled with CellTrace™ Violet proliferation dye) were detached from tissue culture plates using enzyme-free cell dissociation buffer (Thermo Fisher Scientific, Inc.), washed with PBS and resu-pended in PBS containing 2% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA). Sytox AADvanced Dead Cell dye (Thermo Fisher Scientific, Inc.) was used to exclude non-viable cells. FACS was performed using the LSR 11 Flow Cytometer (BD Biosciences), with data analysis performed using FlowJo software (version 7.6.5; FlowJo, LLC, Ashland, OR, USA). Cell proliferation was determined by the extent of CellTrace™ Violet proliferation dye dilution, as determined by FACS analysis. The use of FACS enabled a reliable and efficient, high-throughput analysis of tumor cell proliferation in the tumor co-culture assays, To assess the expression of PECAM-1 on B16-F10 cells and MECs, cells were initially incubated with 1 µg/10⁶ cells anti-mouse CD16/32 for 10 min at 4°C to block non-specific binding to Fc receptors, and were then stained with 10 µg/ml 390 or Mec 13.3 antibodies for 30 min at 4°C and subjected to FACS analysis.

Abraham V., Cao G., Parambath A., Lawal F., Handumrongkul C., Debs R, & Delisser H.M. (2018). Involvement of TIMP-1 in PECAM-1-mediated tumor dissemination. International Journal of Oncology, 53(2), 488-502.

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