Lsrii cell analyzer

Manufactured by BD

Sourced in United States

The BD LSRII Cell Analyzer is a high-performance flow cytometry instrument designed for advanced cellular analysis. The core function of the LSRII is to detect and analyze the physical and fluorescent properties of cells or particles in a liquid sample as they pass through a laser beam.

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BD LSRII analyzer (183 citations) LSRII UV cell analyzer (3 citations)

34 protocols using lsrii cell analyzer

Cell Cycle Analysis by PI Staining

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For cell cycle analysis, we performed propidium iodide (PI) DNA staining followed by flow cytometric analysis. In more detail, 96 hours after siRNA transfection, cells were harvested and used for flow cytometric analysis. Cells were fixed in ice-cold 70% ethanol for 30 min at 4 °C, washed three times with ice-cold PBS and resuspended in 200µl DNA staining solution (PBS containing 50µg/ml PI (Sigma Aldrich, Steinheim, Germany) and 100µg/ml Ribonuclease A (Sigma Aldrich, Steinheim, Germany)). After 15min incubation in dark, cell cycle was analyzed using a LSR II Cell Analyzer (BD Bioscience, Heidelberg, Germany). FCS Express 5 Flow Cytometry software (DeNovoTM Software, Glendale, USA) was used for analyzing flow cytometric data.

Offermann A., Vlasic I., Syring I., Vogel W., Ruiz C., Zellweger T., Rentsch C.A., Hagedorn S., Behrends J., Nowak M., Merseburger A., Bubendorf L., Kirfel J., Duensing S., Adler D, & Perner S. (2016). MED15 overexpression in prostate cancer arises during androgen deprivation therapy via PI3K/mTOR signaling. Oncotarget, 8(5), 7964-7976.

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Quantifying HCV-Induced Apoptosis in Huh-7 Cells

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Huh-7 cells were electroporated with JFH1 RNA, a genotype 2 strain of HCV as previously described (70 (link)). Upon confirming >85% infection rate by HCV NS5A immunofluorescence, Huh-7 cells were plated in 48 well plates. Day 6 Mϕs were spun down onto Huh-7 cells at 400× g for 5 min at a ratio of 2:1. Following 24 h incubation, macrophages were removed by pipetting, and Huh-7 cells detached using Accutase (Sigma-Aldrich). Huh-7 cells were labeled with Annexin V (Becton Dickinson, 550474), Epcam (Miltenyi Biotec, 130-091253) and propidium iodide (Sigma Aldrich, P4864) in 1× Annexin V binding buffer according to the manufacturers protocol. Cells were washed thoroughly, fixed with 2% paraformaldehyde and analyzed on the BD Biosciences LSR II cell analyzer.

Read S.A., Wijaya R., Ramezani-Moghadam M., Tay E., Schibeci S., Liddle C., Lam V.W., Yuen L., Douglas M.W., Booth D., George J, & Ahlenstiel G. (2019). Macrophage Coordination of the Interferon Lambda Immune Response. Frontiers in Immunology, 10, 2674.

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Cell Surface PD-1 Expression Quantification

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Cell surface PD-1 expression was assessed by flow cytometric assays. MIAPaCa-2 and PANC-1 cells were plated on 100 mm³ culture dishes and incubated overnight for cell attachment. The next day, cells were serum-starved overnight. On the 3^rd day, cells were exposed to AMD3100 (1 μM) or solvent control for 45 min. Cells were then lysed and prepared for flow cytometric analysis of PD-1 expression. Mouse anti-PD-1-PE antibody (20 μL per test, BD Biosciences, 557946) was used to probe for cell surface PD-1 expression. Mouse IgG1-PE antibody was used as isotype antibody control and the assay was performed on an LSR II cell analyzer (BD Sciences). Flowjo v10 software was used for flow data analysis.

Harper M.M., Lin M., Cavnar M.J., Pandalai P.K., Patel R.A., Gao M, & Kim J. (2022). Interaction of immune checkpoint PD-1 and chemokine receptor 4 (CXCR4) promotes a malignant phenotype in pancreatic cancer cells. PLoS ONE, 17(7), e0270832.

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Isolation and Analysis of Joint Cells from Arthritic Mice

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Joints from arthritic mice and from mice that had not received arthritogenic serum transfer (nonarthritic controls) were digested using type IV collagenase (1 mg/ml) and DNase I (200 units/ml) in RPMI medium supplemented with 10% fetal calf serum (FCS). Floating cells were recovered from the digestion mix every 20 minutes, and the reaction was stopped by adding ice-cold phosphate buffered saline (PBS)−2% FCS–5mM EDTA. The cells were then spun down for 10 minutes at 1,200 revolutions per minute. Digestion was not carried out longer than 60 minutes.
After a single-cell suspension was obtained, cells were stained with fluorescence-labeled antibodies and then analyzed by flow cytometry. Alternatively, enrichment of CD45− cells that were positive for podoplanin (PDPN+) was achieved by first depleting CD45+ cells using anti-CD45 biotin-labeled antibodies, followed by use of anti-biotin magnetic beads (Miltenyi Biotec). PDPN+ cells were enriched within the population of negatively selected cells with the use of antiphycoerythrin (anti-PE) beads (Miltenyi Biotec) in combination with PDPN–PE labeling. Positively selected cells were harvested, and the purity of the CD45−PDPN+ cell population was assayed by flow cytometry, yielding a purity of ~90%. An LSRII cell analyzer (BD Biosciences) was used for the flow cytometry analyses, and FlowJo software (TreeStar) was used for data analysis.

Garcia-Carbonell R., Divakaruni A.S., Lodi A., Vicente-Suarez I., Saha A., Cheroutre H., Boss G.R., Tiziani S., Murphy A.N, & Guma M. (2016). Critical Role of Glucose Metabolism in Rheumatoid Arthritis Fibroblast-like Synoviocytes. Arthritis & rheumatology (Hoboken, N.J.), 68(7), 1614-1626.

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Cell Apoptosis Assay Using Annexin V-FITC

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Cell apoptosis assays were performed using the annexin V-FITC apoptosis detection kit according to the manufacturer’s protocol. Cells were seeded in 10 cm dishes and treated with 300 nM DAC for 24 h (786-O cells) or 48 h (A-498, A-704, ACHN, Caki-1 and Caki-2). Fresh media were added at days 3 and 6 after treatment. Cells were harvested at day 8 after treatment, stained and analyzed by the LSRII Cell Analyzer (BD Bioscience). The results were analyzed using FlowJo software. Positive controls were generated by incubating cells at 60°C for 20 min. Biological replicates were shown. The percentages of living cell (Q4, Bottom left), and cells undergoing necrosis (Q1, Top left), late apoptosis (Q2, Top right), and early apoptosis (Q3, Bottom right) were plotted for Figure 2D.

Jones T.L., Tucker D.E, & Ort D.R. (1998). Chilling Delays Circadian Pattern of Sucrose Phosphate Synthase and Nitrate Reductase Activity in Tomato. Plant Physiology, 118(1), 149-158.

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Cell Apoptosis Assay Using Annexin V-FITC

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Li H.T., Jang H.J., Rohena-Rivera K., Liu M., Gujar H., Kulchycki J., Zhao S., Billet S., Zhou X., Weisenberger D.J., Gill I., Jones P.A., Bhowmick N.A, & Liang G. (2023). RNA mis-splicing drives viral mimicry response after DNMTi therapy in SETD2-mutant kidney cancer. Cell reports, 42(1), 112016.

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Immune Cell Profiling by Flow Cytometry

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Immune cells were phenotyped by using flow cytometry at baseline and on Days 1, 2, 7, 14, 28, and 56 as previously described [23 (link)] using an LSRII cell analyzer (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo Software (TreeStar, Ashland, OR, USA). All samples were gated to exclude dead cells, debris, and doublets. Dendritic cells (CD3-CD14-CD16-CD19-CD20-CD56-HLA-DR+) were defined as myeloid (mDCs, CD11c+) or plasmacytoid (pDCs, CD123+) and further classified by BDCA1, CCR7, CD86, and CD11b expression. Monocytes (CD19-CD3-HLA-DR+) were subdivided by CD14/CD16 expression and further with CD86 and CCR5. Lymphocytes (CD14−) were divided to T cells (CD3+), B cells (CD19+), and natural killer cells (CD3-CD19-CD56dimCD16+) and further categorized based on CD4 (CD4+ and CD4− for the Leukocyte Panel) and CD8 (for T cell phenotyping), CCR7, CD86, CD11b, HLA-DR, CCR5, CD38, and CD69. Activated T cells were defined by CD38 and HLA-DR co-expression or intracellular co-expression of Ki67 and Bcl-2. CD4 responders were defined at Days 4, 14, or 28 by percentage of CD3+CD4+ CD38+HLA-DR+ effector T cells at least 2 standard deviations (SD) above the mean of all baseline samples. CD8+ T cell responders were defined at Days 7, 14, or 28 by percentage of CD3+CD8+ CD38+HLA-DR+ effector T cells at least 3 SD above the mean of all baseline samples.

Natrajan M.S., Rouphael N., Lai L., Kazmin D., Jensen T.L., Weiss D.S., Ibegbu C., Sztein M.B., Hooper W.F., Hill H., Anderson E.J., Johnson R., Sanz P., Pulendran B., Goll J.B, & Mulligan M.J. (2019). Systems Vaccinology for a Live Attenuated Tularemia Vaccine Reveals Unique Transcriptional Signatures That Predict Humoral and Cellular Immune Responses. Vaccines, 8(1), 4.

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Quantifying hERG Subunit Surface Expression

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Relative surface expression of BBS-tagged hERG subunits was quantitatively determined using quantum dot labeling or used in flow cytometry (using a BD LSRII Cell Analyzer) to determine the BBS-tagged hERG 1a-YFP or hERG 1b-YFP surface density as described previously (Aromolaran et al., 2014 (link)).

Martinez-Mateu L., Saiz J, & Aromolaran A.S. (2019). Differential Modulation of IK and ICa,L Channels in High-Fat Diet-Induced Obese Guinea Pig Atria. Frontiers in Physiology, 10, 1212.

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Assessing Cytotoxicity of ADCs in Breast Cancer Cells

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MDA-MB-468 cells were seeded in 6-well plates at 0.2 to 1 million cells per well in growth medium (DMEM+10%FBS). The following day, medium was removed and replaced with 2 mL fresh growth media containing the following ADCs or a proprietary microtubule inhibitor (PF-06380101): 2 µg/mL RN765C, 2 µg/mL negative control ADC (a non-binding isotype control ADC) or 5 nM PF-06380101. Cells were incubated at 37°C in a humidified CO₂ incubator for 24, 48 or 72 hours. A no treatment control was included in each time point. At the end of treatment, both the medium and cells were harvested and combined into a collection tube. Samples were then processed with CycleTEST Plus DNA Reagent Kit (BD Biosciences) according to manufacturer’s protocol. DNA content data were acquired within 1 hour on a LSRII Cell Analyzer (BD Biosciences). 30,000 events were collected for each sample and cell cycle phase analysis was performed on FlowJo v.10 (FlowJo, LLC).

Wong O.K., Tran T.T., Ho W.H., Casas M.G., Au M., Bateman M., Lindquist K.C., Rajpal A., Shelton D.L., Strop P, & Liu S.H. (2018). RN765C, a low affinity EGFR antibody drug conjugate with potent anti-tumor activity in preclinical solid tumor models. Oncotarget, 9(71), 33446-33458.

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Cell Cycle Analysis of NRP1 Knockdown

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Cells transfected with either scramble control or NRP1 siRNA were washed and fixed in ice-cold 70% ethanol before staining with propidium iodide (50 μg/ml) containing RNase (5 μg/ml) to measure DNA content. Samples were sorted using LSR II Cell Analyzer (BD Biosciences, Franklin Lakes, NJ) for cell-cycle distribution and analyzed using the FCS Express 6 software. A total of 10 000 events were examined per sample.

Bao R., Surriga O., Olson D.J., Allred J.B., Strand C.A., Zha Y., Carll T., Labadie B.W., Bastos B.R., Butler M., Hogg D., Musi E., Ambrosini G., Munster P., Schwartz G.K, & Luke J.J. (2020). Transcriptional analysis of metastatic uveal melanoma survival nominates NRP1 as a therapeutic target. Melanoma Research, 31(1), 27-37.

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