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Facs lsrii instrument

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The BD FACS LSRII is a flow cytometry instrument designed for high-performance multiparameter analysis. It features a compact, modular design and allows for the simultaneous detection of up to 18 parameters. The LSRII provides researchers with a flexible and powerful platform for a wide range of applications in fields such as immunology, cell biology, and biomedical research.

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Lab products found in correlation

Human cd11b apc conjugate, by Thermo Fisher Scientific (2 mentions) Human cd14 pacific blue, by Thermo Fisher Scientific (2 mentions) Collagenase type 1, by Thermo Fisher Scientific (1 mentions) Facsdiva software, by BD (1 mentions) Anti cd19 6d5, by BioLegend (1 mentions) Apc cy7 anti tcr β h57 597, by BioLegend (1 mentions) Percp anti cd45 30 f11, by BD (1 mentions) Pe cy7 anti cd11b m1 70, by BD (1 mentions) Fcr blocking reagent, by Miltenyi Biotec (1 mentions) Methylcellulose, by STEMCELL (1 mentions) Methocult h4434, by STEMCELL (1 mentions) Gentamycin, by Merck Group (1 mentions) Streptavidin apc, by Thermo Fisher Scientific (1 mentions) 24 well plate, by Thermo Fisher Scientific (1 mentions) 2 mercaptoethanol, by Merck Group (1 mentions) Thymidine, by Beckman Coulter (1 mentions) Scintillation counter, by Beckman Coulter (1 mentions) Lung dissociation kit, by Miltenyi Biotec (1 mentions) 96 well microtiter plate, by Thermo Fisher Scientific (1 mentions) 3h thymidine, by PerkinElmer (1 mentions)

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LSRII instrument (326 citations) FACS instrument (23 citations) FACS BD LSRII (12 citations) BD FACSTM (2 citations)

10 protocols using facs lsrii instrument

1

MDS-L Cell Differentiation Assay

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MDS-L cells were seeded in a 6 well plate at a density of 100,000 cells and treated with PRT543 at a concentration of 300nM. Dosing was done starting at Day 0 and continued every third day (total of 7 doses). On Day 21, cells were stained with Human CD11b-APC conjugate (Thermo Fisher Scientific, Waltham, MA, USA, Catalogue No CD11B05, clone VIM12), Human CD14- Pacific blue TM (Thermo Fisher Scientific, Catalogue No MHCD1428, clone Tuk 4). Using a BD FACS LSRII instrument (BD Biosciences, Franklin Lakes, NJ, USA) data was acquired and analyzed using Flow Jo software version 10.6.1 (BD Biosciences).
Venkatasubramanian M., Schwartz L., Ramachandra N., Bennett J., Subramanian K.R., Chen X., Gordon-Mitchell S., Fromowitz A., Pradhan K., Shechter D., Sahu S., Heiser D., Scherle P., Chetal K., Kulkarni A., Myers K.C., Weirauch M.T., Grimes H.L., Starczynowski D.T., Verma A, & Salomonis N. (2024). Broad de-regulated U2AF1 splicing is prognostic and augments leukemic transformation via protein arginine methyltransferase activation. bioRxiv.
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Tumor Immune Profiling by Flow Cytometry

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Flow cytometry analyses were carried out with single cell suspensions prepared from freshly explanted tumors. Tumors were minced with a scalpel. MC38cea tumors were digested for 30 min at 37 °C in RPMI + 5 % FBS + 200 IU/mL Collagenase type I (Thermo Scientific) with vigorous shaking every 10 min. Minced B16hCD46 and digested MC38cea tumor samples were passed through 100 µm cell strainers and cells were pelleted by centrifugation (5 min at 300× g) and counted. 2 × 106 cells were used for staining with the following antibodies (all from BD Biosciences, Heidelberg, Germany): PerCP-Cy5.5 Anti-Mouse CD45.2 (clone 104) 1:500, Alexa Fluor® 700 Anti-Mouse CD3 Molecular Complex (clone 17A2), 1:500, APC-Cy7 Anti-Mouse CD4 (clone GK1.5), 1:500, APC Anti-Mouse CD8a (clone 53–6.7), 1:500, FITC Anti-Mouse CD335 (NKp46) (clone 29A1.4), 1:400, and PE Anti-Mouse CD69 (Clone H1.2F3), 1:250. DAPI staining was used for dead cell exclusion. Data were acquired on a BD FACS LSR II instrument with FACS Diva software (version 8.0.1, BD Biosciences) and analyzed with FlowJo software (version X10.0.7r2, Tree Star Inc., Ashland, OR, USA). Only samples with at least 1000 cells were included in the analysis.
Backhaus P.S., Veinalde R., Hartmann L., Dunder J.E., Jeworowski L.M., Albert J., Hoyler B., Poth T., Jäger D., Ungerechts G, & Engeland C.E. (2019). Immunological Effects and Viral Gene Expression Determine the Efficacy of Oncolytic Measles Vaccines Encoding IL-12 or IL-15 Agonists. Viruses, 11(10), 914.
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3

Murine Immune Cell Profiling

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Single cell suspensions were generated from murine tissues of C57/BL6 and BALB/c mice. Afterwards, cells were treated with FcR-blocking reagent (Miltenyi Biotec) for 10 min at 4°C after washing with flow cytometry buffer. To exclude dead cells, samples were washed twice with PBS and incubated with BD Horizon™ Fixable Viability Stain 510 viability dye (for 10 min at RT in the dark). Subsequently surface staining to discriminate immune cell populations with PerCP anti-CD45 (30-F11, BD Biosciences), APC-Cy 7 anti-TCRβ (H57-597, BioLegend), Pe-Cy7 anti-CD11b (M1/70, BD Biosciences), and anti-CD19 (6D5, BioLegend), was performed. The data was acquired using a BD FACS LSR II instrument (BD Biosciences) and analyzed with FlowJo software (Tree Star Inc, version 9.9.6 or 10.4.1).
Schreiber L., Ghimire S., Hiergeist A., Renner K., Althammer M., Babl N., Peuker A., Schoenhammer G., Hippe K., Gessner A., Albrecht C., Pielmeier F., Büttner-Herold M., Bruns H., Hoffmann P., Herr W., Holler E., Peter K., Kreutz M, & Matos C. (2024). Strain specific differences in vitamin D3 response: impact on gut homeostasis. Frontiers in Immunology, 15, 1347835.
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4

Clonogenic Assays for Myeloid Differentiation

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Primary patient MDS samples were plated in Methylcellulose (Stem cell technologies, H4435, Vancouver, CA) with PRT543 at different concentrations and control and colonies were counted after 14 −17 days. This was followed by staining and processing by Flow cytometry (BD FACS LSRII instrument) for Erythroid and Myeloid differentiation. Antibodies used were Human CD45 PE-Cy7; Human CD34 PE; Human Gly-A PerCP-Cy5.5; Human CD14 Pacific blue; Human CD71 FITC and Human CD11b APC. For THP1 and MDSL clonogenic assays, clonogenic frequencies were determined by plating cell lines in Methocult H4434 (StemCell Technologies) in SmartDish meniscus-free 6-well plates (StemCell Technologies). Plates were kept in humidified chambers and colonies were imaged and manually scored after 9–14 days using the STEMvision counter (StemCell Technologies).
Venkatasubramanian M., Schwartz L., Ramachandra N., Bennett J., Subramanian K.R., Chen X., Gordon-Mitchell S., Fromowitz A., Pradhan K., Shechter D., Sahu S., Heiser D., Scherle P., Chetal K., Kulkarni A., Myers K.C., Weirauch M.T., Grimes H.L., Starczynowski D.T., Verma A, & Salomonis N. (2024). Broad de-regulated U2AF1 splicing is prognostic and augments leukemic transformation via protein arginine methyltransferase activation. bioRxiv.
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5

Characterization of D1 Myeloid Dendritic Cells

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The D1 cell line, a long-term growth factor-dependent immature myeloid (CD11b+, CD8α-) DC line of splenic origin derived from a female C57BL/6 mouse, was generously provided by prof. P. Ricciardi-Castagnoli (University of Milan-Bicocca, Italy) (39 (link)). The D1 cells were cultured in 24-well plates (Nunc, A/S Roskilde, Denmark) in Iscove's medium (Biochrom KG, Germany), supplemented with 10% heat-inactivated fetal calf serum (Biochrom KG, Germany), 50 μM 2-mercaptoethanol (Sigma Aldrich, Sweden), 1 mM L-glutamine (Biochrom KG, Germany) and 50 μg/ml Gentamycin (Sigma Aldrich, Sweden) and stimulated for different times with 0. 2 μM of CTA1-3Eα-DD soluble protein or when incorporated into NPLs. To assess the processing efficiency of fusion protein we determined the cell surface expression of peptide plus MHC II complex by incubating D1 cells with anti-Eα(52-68):I-Ab complex-specific Y-Ae biotin-labeled antibody (eBiosciences, USA). Flow cytometric analysis was performed after incubation with streptavidin-APC and anti-CD11c-PE, 7AAD, MHCII-FITC at 4°C for 30 min (eBiosciences, USA). We analyzed 100,000 events using a BD-FACS LSR II instrument (BD Bioscience, USA) and the data were analyzed with FlowJo (TreeStar, USA) software.
Bernasconi V., Bernocchi B., Ye L., Lê M.Q., Omokanye A., Carpentier R., Schön K., Saelens X., Staeheli P., Betbeder D, & Lycke N. (2018). Porous Nanoparticles With Self-Adjuvanting M2e-Fusion Protein and Recombinant Hemagglutinin Provide Strong and Broadly Protective Immunity Against Influenza Virus Infections. Frontiers in Immunology, 9, 2060.
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6

Analyzing M2e-specific CD4+ T cells

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Two weeks after challenge, lung cells were dissociated using Lung Dissociation Kit (Miltenyi Biotec Norden AB, Sweden), according to the manufacturer’s instructions. To assess the presence of M2e-specific CD4+ T cells, lung cells were stained with M2e-tetramer-PE (NIH Tetramer core facility, USA) at 37 °C for 30 min and then incubated with fluorescently labeled antibodies for surface antigens at 4 °C for 30 min (all from eBiosciences, USA). In all, 1,000,000 events were acquired on a BD-FACS LSR II instrument (BD Bioscience, USA) and data analyzed with FlowJo (TreeStar, USA) software. Thresholds of positivity were identified using fluorescence minus one control for each color on cells from each sample group. For proliferation analysis, single cell suspensions (2 × 106 cells/ml) from lung from immunized and control mice were cultured for in vitro M2e-peptide recall responses in triplicates in 96-well microtiter plates (Nunc, Denmark) for 72 h at 37 °C in 5% CO2. After 72 h, we added [3 H]-thymidine (PerkinElmer, USA) to the cultures for the last 6 h and [3 H]-thymidine uptake was determined using a scintillation counter (Beckman, Sweden).
Schussek S., Bernasconi V., Mattsson J., Wenzel U.A., Strömberg A., Gribonika I., Schön K, & Lycke N.Y. (2020). The CTA1-DD adjuvant strongly potentiates follicular dendritic cell function and germinal center formation, which results in improved neonatal immunization. Mucosal Immunology, 13(3), 545-557.
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7

Flow Cytometric Analysis of Lymphoid Cells

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Lymph nodes were forced through a nylon mesh using a syringe plunger. Peyer’s patches were washed with 2 mM EDTA for 30 min at 37 °C and then digested with Collagenase D (0.5 U/ml final) for 1 h at 37 °C. Supernatant and remaining tissue pieces were forced through a nylon mesh using a syringe plunger. Cells were stained with fixable viability die Aqua for 30 min at 4 °C, washed and then incubated with fluorescently labeled antibodies for surface antigens for 30 min at 4 °C (see Supplementary Table 1). Two separate panels were used for detection of GC B cells and TFH cells, respectively. Intracellular labeling for FoxP3 and Bcl-6 transcription factors was performed after fixation of cells for 30 min at 4 °C using the FoxP3 Transcription Factor Fixation/Permeabilization kit (eBioscience, USA). After labeling, cells were washed twice and analyzed using a BD-FACS LSR II instrument (BD Bioscience, USA) or a CytoFLEX flow cytometer (Beckman Coulter, USA) and data analyzed with FlowJo (TreeStar, USA) software.
Schussek S., Bernasconi V., Mattsson J., Wenzel U.A., Strömberg A., Gribonika I., Schön K, & Lycke N.Y. (2020). The CTA1-DD adjuvant strongly potentiates follicular dendritic cell function and germinal center formation, which results in improved neonatal immunization. Mucosal Immunology, 13(3), 545-557.
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8

Myeloid Differentiation and Apoptosis Assay

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Myeloid differentiation: ASXL1 mutant and corrected KBM5 cells were stained with Human CD11b–APC Conjugate (Thermo Fisher Scientific, Waltham, MA, USA, Cat No. CD11B05, clone VIM12), Human CD14-Pacific BlueTM (Thermo Fisher Scientific, Cat No. MHCD1428, clone TuK4), and Anti-Human CD15-PerCP-eFluorTM710 (Thermo Fisher Scientific, Cat No. 46-0158-42, clone MMA). Data acquisition was performed using a BD FACS LSRII instrument (BD Biosciences, Franklin Lakes, NJ, USA) and data were analyzed using FlowJo software version 10.6.1 (BD Biosciences). Apoptosis: ASXL1 mutant and corrected KBM5 cells were treated with 40 nM of VEN for 48 h with controls. At hour 48, cells were stained with FITC-conjugated Annexin V Alexa Fluor 488 (Thermo Fisher Scientific, Cat No. A13199) and Propidium iodide, and flow cytometry was conducted using a BD FACS LSRII instrument to evaluate the percentage of viable cells, the percentage of cells in early and late apoptosis, and cells undergoing apoptosis. Data were analyzed using FlowJo.
Rahmani N.E., Ramachandra N., Sahu S., Gitego N., Lopez A., Pradhan K., Bhagat T.D., Gordon-Mitchell S., Pena B.R., Kazemi M., Rao K., Giricz O., Maqbool S.B., Olea R., Zhao Y., Zhang J., Dolatshad H., Tittrea V., Tatwavedi D., Singh S., Lee J., Sun T., Steidl U., Shastri A., Inoue D., Abdel-Wahab O., Pellagatti A., Gavathiotis E., Boultwood J, & Verma A. (2021). ASXL1 mutations are associated with distinct epigenomic alterations that lead to sensitivity to venetoclax and azacytidine. Blood Cancer Journal, 11(9), 157.
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9

Measuring Integrin Activation in CHO Cells

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Activation of integrin αIIbβ3 was measured by PAC-1 mAb specifically recognizing activated αIIbβ3 integrin (Becton Dickinson Immunocytometry System) as described previously (Chang et al., 2014 (link); Yang et al., 2014 (link)). Briefly, the CHO-A5 cells stably expressing integrin αIIbβ3 were transfected with GFP or GFP-tagged talin or mutant talin using Lipofectamine 2000 (Invitrogen). One day after transfection, the cells were resuspended in Tyrode’s buffer and incubated with PAC-1 mAb for 1 hr at 4°C. After three washes with Tyrode’s buffer, Alexa Fluor 647 labeled secondary antibody was added to the cells for 1 hr on ice. Stained cells were analyzed using an LSRII FACS instrument (BD Scientific). The data were processed by FlowJo software. Histogram analyses of the relative PAC1 binding were performed by gating cells with GFP fluorescence intensity (FL unit) between 5×104 to 1×105 unless otherwise specified. The mean fluorescence intensity (MFI) of the cells transfected with empty GFP vector was defined as 1 and all other constructs were compared with the GFP control. The data are shown as the means ± SD from three experiments repeated under the same conditions. Unpaired t test was determined by GraphPad software to calculate the P value. Talin were examined by Western blot to ensure equal expression levels in CHO-A5 cells (Fig. S2B).
Zhang H., Chang Y.C., Huang Q., Brennan M.L, & Wu J. (2016). Structural and functional analysis of a talin triple domain module suggests an alternative talin autoinhibitory configuration. Structure (London, England : 1993), 24(5), 721-729.
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10

Profiling Secretome of Senescent Cells

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Cells were washed, seeded at 1 × 106/well for CTRL cells or – to omit confluence – at 5 × 105/well for drug-treated senescent cells in 6 well plates. At day 1, medium was removed and 1.5 ml fresh medium/106 cells was added. After 24 h the supernatant was collected, cellular components were removed by centrifugation and supernatants were snap-frozen. Supernatants were analyzed by G4000 protein array at Raybiotech (USA) for 274 secreted factors. Further analysis was done in R using Bioconductor packages and custom scripts. Background corrected .gpr files were normalized to the internal control and genes with higher than 2-fold difference in the HU, CPT or BrdU-treated senescent NB-cells compared to control were selected for visualization.
Qantitative analysis was carried out by ELISA (MCP-3, Abnova, Taiwan), AlphaLISA (MMP-9, IL-6, RANTES; PerkinElmer, Austria) or Flowcytomix (OPG, VEGFA, PDGF-AA; eBioscience, Austria) according to the manufacturer's instructions. For detection a plate reader (Enspire, PerkinElmer) or the LSRII FACS instrument (BD) was used, respectively.
Taschner-Mandl S., Schwarz M., Blaha J., Kauer M., Kromp F., Frank N., Rifatbegovic F., Weiss T., Ladenstein R., Hohenegger M., Ambros I.M, & Ambros P.F. (2015). Metronomic topotecan impedes tumor growth of MYCN-amplified neuroblastoma cells in vitro and in vivo by therapy induced senescence. Oncotarget, 7(3), 3571-3586.
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