Spleens or lymph nodes were crushed into single cell suspensions in cold 1x PBS. Bone marrow cells were harvested from femurs by flushing with 1x PBS. Cells were washed and red blood cells (RBCs) lysed with 0.165M NH4Cl. 106 cells per sample were labeled with a live/dead dye and Fc receptors blocked with 2.4G2 before Ab staining. Lymphocytes and monocytes were identified by forward and side-scatter profiles. GC B cells were identified as CD19+ GL7+ FAS+; Tfh cells as CD4+ PD-1+ CXCR5+; ASCs as CD138+ B220lo/neg; DCs as CD11chi CD19− CD3− CD49b− Ter119− and CD8α+ or CD8α−; pDCs as CD11c+ CD19− CD3− CD49b− Ter119− CD45RA+; eosinophils as Siglec-F+ CD11bint. For intracellular cytokine analysis, cells were fixed and permeabilized (BD Biosciences) overnight at 4°C before staining. For intracellular IL-12, cells were incubated for 3 hours at 37°C with 3 ug/ml Brefeldin A before fixing. For chemokine receptor analysis, cells were labeled in azide-free buffer at 37°C, then washed in normal buffer on ice to halt recycling. Data for DC analyses were collected on a Becton Dickinson LSR II and analyzed with FlowJo. All other samples were run on a Beckman Coulter CyAn ADP and analyzed with Dako Summit.
Lsr 2
Manufactured by FlowJo
Sourced in United States
The LSR II is a flow cytometer designed for high-performance cell analysis and sorting. It offers a range of advanced features and capabilities to support a variety of research applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto, by FlowJo (4 mentions)
Anti cd16 32, by BD (3 mentions)
Facscanto 2, by FlowJo (3 mentions)
Alexa fluor 594 anti vimentin clone epr3776, by Abcam (2 mentions)
Apc anti cd31 clone 390, by Thermo Fisher Scientific (2 mentions)
Fitc anti ly 6c clone al 21, by BD (2 mentions)
Pe cy5 labeled anti cd5 clone 53 7, by BioLegend (2 mentions)
Pe cy7 anti gr 1 clone rb6 8c5, by Thermo Fisher Scientific (2 mentions)
Apc cy7 anti cd11b clone m1 70, by BioLegend (2 mentions)
Pacific blue anti f4 80 clone bm8, by BioLegend (2 mentions)
Perm buffer 3, by Thermo Fisher Scientific (2 mentions)
Phosflow fix buffer 1, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Oct4 c 10, by Santa Cruz Biotechnology (2 mentions)
Sox2 57ct23, by Abcam (2 mentions)
Nanog h 155, by Santa Cruz Biotechnology (2 mentions)
Hoxb4 ep1919y, by Abcam (2 mentions)
Facscalibur, by FlowJo (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Bodipy fl c16, by Thermo Fisher Scientific (2 mentions)
37 protocols using lsr 2
1
Comprehensive Immune Cell Profiling
2
Comprehensive Flow Cytometry Analysis of T Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies were purchased from eBioscience (San Diego, California, USA), Biolegend (San Diego, California, USA) and BD Biosciences (Mississauga, Ontario, Canada). Vγ6-specific staining was accomplished in some experiments using undiluted supernatants from the 17D1 hybridoma (Sunnybrook Core Antibody Facility)66 (link). A negative gating strategy was used in most experiments to identify Vγ6+ T cells, as previously described67 (link) and shown in Fig. 7b . Cells were washed and incubated with Fc blocking antibody (clone 2.4G2, Sunnybrook Core Antibody Facility, 1 mg/ml), followed by extracellular staining for surface CD4 (clone GK1.5), CD8α (clone 53–6.7), CD3 (clone 145-2C11), TCRγδ (clone GL3), TCRβ (clone Η57–597), Vγ4 (Vγ2; clone UC3-10A6), Vγ5 (Vγ3; clone 536), Vγ1 (Vγ1.1; clone 2.11), Vγ7 (clone F2.67), CD27 (clone LG3-1A10), CD24 (clone M1/69) and/or CD73 (clone TY11.8). All flow cytometric analyzes were performed with a Becton-Dickenson (BD) LSRII, Diva software, and FlowJo software. Sorting was performed on a FACSARIA (BD). Representative gating strategies are shown in Supplementary Fig. 11 .
3
Flow Cytometry Analysis of Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All antibodies for flow cytometry for DC markers and intracellular staining were obtained from BD Bioscience (Mississauga, Canada). They included APC-conjugated rat anti-mouse CD11a, PE-CY7-conjugated hamster anti-mouse CD11c, PE-conjugated rat anti-mouse CD40, FITC-conjugated rat anti-mouse I-Ab, APC-conjugated rat anti-mouse IL-12, IL-6 and TNF-α, and their respective isotype control antibodies. For DCs staining, cells were pre-incubated with Fc receptor blocker for 10 min at room temperature (RT), and then stained with antibodies against CD11b, CD11c, CD40 and MHCII in PBS containing 0.2% BSA. Labeled cells were run on a FACSCanto or LSRII flow cytometer and data were analyzed by FlowJo (v. 7.15) software.
4
Cardiac Fibroblast Isolation and Cell Cycle Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cardiac fibroblasts were isolated from hearts at 1 wk post-MI by langendorff perfusion. GFP-positive cells were gated for analysis and DAPI were used for analyzing DNA content. FACS were performed on BD Biosciences SORP Aria I and BD Biosciences LSRII and cell cycle modeling were processed with FlowJo software.
5
Cell Cycle Analysis of Osteosarcoma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All OS cell lines were analysed for cell cycle progression using Bromodeoxyuridine (BrdU) and Propidium iodide (PI) staining as previously described [34 (link)]. Briefly, OS cells were incubated for 72 h with medium containing vehicle or GIC50 CX-5461 concentrations (determined from the cell proliferation assay), then labelled with 10 µM BrdU (Sigma-Aldrich, St. Louis, MO, USA), harvested and stained with PI. Flow cytometry (Becton Dickinson LSR II) was used to assess the level of staining, and FlowJo software (version 10.0) was used for data analysis.
6
Multiparametric Flow Cytometry of Aortic and Splenic Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single cell suspensions from the aortae and spleen were obtained as described above and incubated with anti-CD16/32 (BD Biosciences, 553142) and stained on ice for 30 minutes with the following antibodies: Alexa Fluor 594-anti-Vimentin (clone EPR3776, Abcam, ab154207), APC-anti-CD31 (clone 390, Invitrogen, 17–0311-80), FITC-anti-Ly-6C (clone AL-21, BD Biosciences, 553104), PE-Cy5-labeled anti-CD5 (clone 53–7.3, BioLegend, 100609), PE-Cy7-anti-Gr-1 (clone RB6–8C5, Invitrogen, 25–5931-81), APC-Cy7-anti-CD11b (clone M1/70, BioLegend, 101225), and Pacific Blue-anti-F4/80 (clone BM8, BioLegend, 123123). For intracellular staining, cells were fixed and permeabilized with buffers (BD Phosflow Fix Buffer I and Perm Buffer III) according to the manufacturer’s instructions, then stained with Alexa Fluor 488-anti-alpha-smooth muscle actin (α-SMA, clone 1A4, eBioscience, 50–112-4644). Cell suspensions were subjected to flow cytometry (Becton Dickinson LSR II) and analyzed using FlowJo10.1.r5. Macrophages were identified as CD11b+/Ly-6Clow/F4/80+ cells. Ly-6Chi monocytes were identified as CD11b+/Ly-6Chi/F4/80low cells. Neutrophils were identified as CD11b+/Gr-hi cells.
7
Multi-Marker Immunophenotyping of T-Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prevent unspecific binding of mAbs, all samples were pre incubated with 25 µL of Fc block at 4°C for 10 min. Surface staining was carried out for 20 min at 4°C using Allophycocyanin (APC)-labeled anti-CD4 (clone RM4-5; BD Heidelberg, Germany) or Brilliant Violet 510-labeled anti-CD4 (clone RM4-5, Biolegend), Phycoerythrin (PE)-labeled anti-CD 304 (Neuropilin-1, clone 3E12, Biolegend), PE- or FITC-labeled anti-CD103 (clone 2E7, Biolegend), Peridinin chlorophyll protein-cyanine5-labeled anti-CD11b (clone M1/70, BD, Heidelberg, Germany.) and PE-labeled anti-Gr-1 (clone 1A8, BD). For intracellular staining cells were permeabilized with 250 µl fixation/permeabilization buffer for 30 min at 4°C, washed with permeabilization buffer and stained with PE- or Alexa Fluor 700-labeled anti-Foxp3 (clone FJK-16s, eBiosciences, SanDiego, USA) and APC–labeled anti-Helios (clone 22F6, Biolegend). For analysis of Neuropilin and Helios expression cells were also stained for 30 min on ice with LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV excitation according to the manufacturers recommendation (Life Technologies, Darmstadt, Germany). Samples were measured on a BD FACSCalibur or LSRII and analyzed using FlowJo software.
8
Phenotypic and Functional Analysis of Human Hematopoietic Stem and Progenitor Cells
9
Multiparametric Flow Cytometry of Aortic and Splenic Immune Cells
10
Cytometric Analysis of Lymph Nodes and Footpad Lesions
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!