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Lsrii 5 laser flow cytometer

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The BD LSRII 5-laser flow cytometer is a high-performance instrument designed for advanced flow cytometry applications. It features five solid-state lasers that enable the simultaneous detection and analysis of up to 18 parameters. The LSRII is capable of performing complex multicolor experiments and provides researchers with the tools needed to conduct in-depth cellular analysis.

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Lab products found in correlation

Facsdiva v6, by BD (2 mentions) Fitc annexin 5 apoptosis detection kit, by BD (2 mentions) Dq ovalbumin, by Thermo Fisher Scientific (2 mentions) Antibodies against gfp and ia ie af647, by BioLegend (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Perm buffer 3, by BD (1 mentions) Fixation permeabilization solution kit, by BD (1 mentions) Cytofix buffer, by BD (1 mentions) Dreg 56, by BD (1 mentions) Vβ11 c21, by Beckman Coulter (1 mentions) Mq1 17h12, by BD (1 mentions) Anti cd8 percp cy5, by BD (1 mentions) Anti cd4 apc alexafluor750, by Beckman Coulter (1 mentions) Alexa fluor 647 antibody labeling kit, by Thermo Fisher Scientific (1 mentions) Transcription factor buffer set, by BD (1 mentions) Cytoflex flow cytometer, by Beckman Coulter (1 mentions) Aqua live dead dye, by Thermo Fisher Scientific (1 mentions) Ebioscience ic fixation buffer, by Thermo Fisher Scientific (1 mentions) Collagenase type 1 c, by Avantor (1 mentions) Countbright beads, by Thermo Fisher Scientific (1 mentions)

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LSRII flow cytometer (4014 citations) LSRII cytometer (559 citations) 5-laser LSRII (19 citations)

16 protocols using lsrii 5 laser flow cytometer

1

Maintenance and Analysis of Neuroblastoma Cell Lines

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Human neuroblastoma cell lines (NGP, SH-SY5Y, IMR-32, CHLA-255) and mouse NB cell line, NB975 were routinely maintained and cultured as described previously (11 (link)). For the in vitro assays performed in the present study, cells were cultured routinely and then partially starved for at least 16-24 hours by culturing in medium supplemented with 1% FBS unless otherwise stated, before treating with drugs or cytokine. Primary antibodies anti-CD114-PE (554538), anti-CD56-APC (555518), anti-CD24-FITC (553261) and fluorochrome, isotype-matching mAbs for negative controls were purchased from BD Biosciences. DAPI (4′,6-diamidino-2-phenylindole, Invitrogen, D3571) staining was used to exclude dead cells in all flow cytometry analysis. LSRII 5-laser flow cytometer (BD Biosciences) was used to perform flow cytometry experiments followed by analysis using FlowJo vX.0.7 (TreeStar). All flow cytometry assays were performed with antibody isotype controls. Stattic (S7947), etoposide (VP-16, E1383) from Sigma-Aldrich and recombinant human G-CSF (BP000175-GD16) was purchased from Syd Labs, Natick, MA. Anti-G-CSF antibody (MAB414) and matching isotype antibody control (MAB005) was purchased from R&D systems, Minneapolis, MN.
Agarwal S., Lakoma A., Chen Z., Hicks J., Metelitsa L.S., Kim E.S, & Shohet J.M. (2015). G-CSF promotes neuroblastoma tumorigenicity and metastasis via STAT3-dependent cancer stem cell activation. Cancer research, 75(12), 2566-2579.
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2

Evaluating Apoptosis in Neuroblastoma Cells

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Cell apoptosis was evaluated in NGP, SH-SY5Y, LAN-5, LAN-5 si-p53, and SK-N-AS neuroblastoma cells treated with RG7388 with the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, San Jose, CA, USA, Ca 556547) per recommended protocol.
FITC Annexin V expression was measured by flow cytometry conducted on an LSR II 5-laser flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with BD FACSDiva v6.1.2 (BD Biosciences).
Lakoma A., Barbieri E., Agarwal S., Jackson J., Chen Z., Kim Y., McVay M., Shohet J.M, & Kim E.S. (2015). The MDM2 small-molecule inhibitor RG7388 leads to potent tumor inhibition in p53 wild-type neuroblastoma. Cell Death Discovery, 1, 15026-.
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3

Placental Macrophage Death Assay

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Placental macrophages were plated, rested, and exposed to glucose or the metabolic stress components as they were for cytokine analysis above. Treatments were performed for 4 or 24 h before cells were harvested for flow cytometric analysis. Cellular death was assayed by dual-staining with annexin V-AF 488 and propidium iodide (PI) following manufacturer’s instructions. Briefly, some PMs were stained with anti-human CD68 to assess purity before being stained with the annexin/PI staining kit. Paraformaldehyde-fixed placental macrophages were acquired immediately after staining on an LSRII 5-laser flow cytometer (BD Biosciences). Living cells were defined as PI-/annexin-; necrotic cells were defined as PI+/annexin-; early apoptotic cells were defined as PI-/annexin+; and late apoptotic or pyroptotic cells were defined as PI+/annexin+. Data files were analyzed using BD Diva (BD Biosciences).
Rogers L.M., Serezani C.H., Eastman A.J., Hasty A.H., Englund-Ögge L., Jacobsson B., Vickers K.C, & Aronoff D.M. (2019). Palmitate induces apoptotic cell death and inflammasome activation in human placental macrophages. Placenta, 90, 45-51.
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4

Evaluating Apoptosis in Neuroblastoma Cells

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Cell apoptosis was evaluated in NGP, SH-SY5Y, LAN-5, LAN-5 si-p53, and SK-N-AS neuroblastoma cells treated with RG7388 with the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, San Jose, CA, USA, Ca 556547) per recommended protocol.
FITC Annexin V expression was measured by flow cytometry conducted on an LSR II 5-laser flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with BD FACSDiva v6.1.2 (BD Biosciences).
Lakoma A., Barbieri E., Agarwal S., Jackson J., Chen Z., Kim Y., McVay M., Shohet J.M, & Kim E.S. (2015). The MDM2 small-molecule inhibitor RG7388 leads to potent tumor inhibition in p53 wild-type neuroblastoma. Cell Death Discovery, 1, 15026-.
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5

Immunophenotyping of GD2.CAR T Cells

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Immunophenotyping was performed using the monoclonal antibodies (mAbs) and reagents detailed in Supplemental Methods. GD2.CARs were detected with the 14G2a anti-idiotype 1A7 mAb. Analysis was performed on an LSR-II 5-laser flow cytometer (BD Biosciences) using BD FACSDiva software version 6.0 and FlowJo 10.1 (Tree Star, Ashland, OR). SPICE software was used to evaluate expression of exhaustion markers (26 (link)).
Xu X., Huang W., Heczey A., Liu D., Guo L., Wood M., Jin J., Courtney A.N., Liu B., Di Pierro E.J., Hicks J., Barragan G.A., Ngai H., Chen Y., Savoldo B., Dotti G, & Metelitsa L.S. (2019). NKT cells co-expressing a GD2-specific chimeric antigen receptor and IL-15 show enhanced in vivo persistence and antitumor activity against neuroblastoma.. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(23), 7126-7138.
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6

NKT-cell Phenotyping and Characterization

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NKT-cell phenotype was assessed using monoclonal antibodies (mAbs) for CD3 (UCHT1), Vα24-Jα18 (6B11), CD4 (RPA-T4), granzyme B (GB11), CD62L (DREG-56; BD Biosciences, San Jose, CA), Vβ11 (C21; Beckman Coulter, Brea, CA), and IL-21R (17A12; BioLegend, ‎San Diego, CA and BD Biosciences). CD19-CAR expression by transduced NKTs was detected using anti-Id mAb (clone 136.20.1) (25 (link)), a gift from Dr. B. Jena (MD Anderson Cancer Center, Houston, TX). Intracellular staining was performed using a fixation/permeabilization solution kit (BD Biosciences) with mAbs for Bcl2 (N46–467; BD Biosciences) and BIM (Y36; Abcam, Cambridge, MA) followed by staining with a secondary goat anti-rabbit IgG-AF488 mAb (Abcam). Phosflow staining was performed using Cytofix buffer (BD Biosciences) and Perm buffer III (BD Biosciences) with mAb for Stat3 (pY705; Clone 4; BD Biosciences). Detection of Stat3 phosphorylation was performed after 15 minutes of treatment with IL-21. Fluorochrome- and isotype-matching antibodies suggested by BD Biosciences or R&D Systems were used as negative controls. Analysis was performed on an LSR-II 5-laser flow cytometer (BD Biosciences) using BD FACSDiva software version 6.0 and FlowJo 10.1 (Tree Star, Ashland, OR).
Ngai H., Tian G., Courtney A.N., Ravari S.B., Guo L., Liu B., Jin J., Shen E.T., Di Pierro E.J, & Metelitsa L.S. (2018). IL-21 selectively protects CD62L+ NKT cells and enhances their effector functions for adoptive immunotherapy. Journal of immunology (Baltimore, Md. : 1950), 201(7), 2141-2153.
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7

Cytokine Responses in Infant Immunity

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Whole blood cytokine levels and intracellular cytokine staining were conducted on heparinized blood drawn from 10-week old South African infants. 10 milliliters of blood were taken and used both for the cytokine assays and genotyping. Samples were stimulated ex-vivo with media or BCG (strain SSI, 1.2 x 106 CFU’s/ml) for 7 hours at 37°C. For whole-blood assays, IL-2 and IFN- γ were measured by multiplex bead array technology according to manufacturer’s instructions (Bio-Rad, Hercules, CA, USA) and read on a Luminex luminometer (Luminex, Austin, TX, USA). Basal cytokine levels measured in plasma harvested from unstimulated blood were subtracted from values obtained from BCG-stimulated blood [11 (link)].
For intracellular cytokine staining, Brefeldin A was added to the above samples and cells were incubated for 5 additional hours. Samples were frozen in 40% RPMI and 40% DMSO with 10% FCS and thawed prior to the time of data analysis. Flow cytometry was performed at the University of Washington Flow Cytometry Core Facility on an LSRII 5-laser flow cytometer (Becton Dickenson, Inc) using antibodies against cell surface markers including: anti-CD3 ECD (Beckman Coulter, UCHT1), anti-CD4 APC-Alexa Fluor 750 (Beckman Coulter, 13B8.2), and anti-CD8 PerCP Cy5.5 (BD, SK1). Antibodies against cytokines included anti-IL-2 PE (BD, MQ1-17H12) and anti-IFN-γ (BD, B27) [33 (link)].
Graustein A.D., Misch E.A., Musvosvi M., Shey M., Shah J.A., Seshadri C., Aguoju A., Bowman K., Mulenga H., Veldsman A., Hanekom W.A., Hatherill M., Scriba T.J, & Hawn T.R. (2018). Toll-like receptor chaperone HSP90B1 and the immune response to Mycobacteria. PLoS ONE, 13(12), e0208940.
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8

TLR2 Expression in U937 Cells

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Wild type and HSP90B1 knockdown U937 cell lines were incubated with either mouse anti human TLR2 conjugated to APC (eBioscience 17–9922) or isotype control. Flow cytometry was performed at the University of Washington Flow Cytometry Core Facility on an LSRII 5-laser flow cytometer (Becton Dickenson, Inc).
Graustein A.D., Misch E.A., Musvosvi M., Shey M., Shah J.A., Seshadri C., Aguoju A., Bowman K., Mulenga H., Veldsman A., Hanekom W.A., Hatherill M., Scriba T.J, & Hawn T.R. (2018). Toll-like receptor chaperone HSP90B1 and the immune response to Mycobacteria. PLoS ONE, 13(12), e0208940.
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9

Antigen Uptake in Intestinal Organoids

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Organoids were formed and cultured for one week in Matrigel and Mouse Intesticult media. They were collected and reseeded without Matrigel in media with 100 μg/mL DQ-Ovalbumin (Thermo Fisher Scientific) for approximately 24 hours. After 24 hours, organoids were fixed, stained overnight with antibodies against GFP and Ia/Ie-AF647 (1:100; Biolegend), and analyzed using a BD LSRII 5-laser flow cytometer. Flow data were analyzed using Cytobank (Kotecha et al., 2010 ).
Chen B., Scurrah C.R., McKinley E.T., Simmons A.J., Ramirez-Solano M.A., Zhu X., Markham N.O., Heiser C.N., Vega P.N., Rolong A., Kim H., Sheng Q., Drewes J.L., Zhou Y., Southard-Smith A.N., Xu Y., Ro J., Jones A.L., Revetta F., Berry L.D., Niitsu H., Islam M., Pelka K., Hofree M., Chen J.H., Sarkizova S., Ng K., Giannakis M., Boland G.M., Aguirre A.J., Anderson A.C., Rozenblatt-Rosen O., Regev A., Hacohen N., Kawasaki K., Sato T., Goettel J.A., Grady W.M., Zheng W., Washington M.K., Cai Q., Sears C.L., Goldenring J.R., Franklin J.L., Su T., Huh W.J., Vandekar S., Roland J.T., Liu Q., Coffey R.J., Shrubsole M.J, & Lau K.S. (2021). Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps. Cell, 184(26), 6262-6280.e26.
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10

Antigen Uptake in Intestinal Organoids

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Organoids were formed and cultured for one week in Matrigel and Mouse Intesticult media. They were collected and reseeded without Matrigel in media with 100 μg/mL DQ-Ovalbumin (Thermo Fisher Scientific) for approximately 24 hours. After 24 hours, organoids were fixed, stained overnight with antibodies against GFP and Ia/Ie-AF647 (1:100; Biolegend), and analyzed using a BD LSRII 5-laser flow cytometer. Flow data were analyzed using Cytobank (Kotecha et al., 2010 ).
Chen B., Scurrah C.R., McKinley E.T., Simmons A.J., Ramirez-Solano M.A., Zhu X., Markham N.O., Heiser C.N., Vega P.N., Rolong A., Kim H., Sheng Q., Drewes J.L., Zhou Y., Southard-Smith A.N., Xu Y., Ro J., Jones A.L., Revetta F., Berry L.D., Niitsu H., Islam M., Pelka K., Hofree M., Chen J.H., Sarkizova S., Ng K., Giannakis M., Boland G.M., Aguirre A.J., Anderson A.C., Rozenblatt-Rosen O., Regev A., Hacohen N., Kawasaki K., Sato T., Goettel J.A., Grady W.M., Zheng W., Washington M.K., Cai Q., Sears C.L., Goldenring J.R., Franklin J.L., Su T., Huh W.J., Vandekar S., Roland J.T., Liu Q., Coffey R.J., Shrubsole M.J, & Lau K.S. (2021). Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps. Cell, 184(26), 6262-6280.e26.
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