Isolated PBMCs were washed with PBS and centrifuged for 5 min at 500 g. The supernatant was discarded and the cells were resuspended in PBS to a cell density of 1×106 cells/100 µl. Cell surface staining was performed on PBMCs using the following anti-human monoclonal antibodies: CD127-AF647 (1:100; clone HIL-7R-M21, cat. no. 558598), CD4-v450 (1:500; clone RPA-T4, cat. no. 560346), CD25-APC-cy7 (1:100; clone M-A251, cat. no. 561782) and TIGIT-PE (1:300; clone 1G9, cat. no. 565168). All antibodies were purchased from BD Pharmingen™. All data were collected on a FACS Aria III™ (BD Biosciences) and were analyzed with Flow Jo software version 7.5 (Tree Star, Inc.).
Flow jo software version 7
Manufactured by Tree Star
Sourced in United States
FlowJo software version 7.5 is a data analysis tool for flow cytometry applications. It provides a user-friendly interface for visualizing and analyzing data from flow cytometers. The core function of FlowJo 7.5 is to enable researchers to manage, analyze, and present their flow cytometry data effectively.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 3, by BD (1 mentions)
Facscan flow cytometer, by Tree Star (1 mentions)
Cd20 efluor 450, by Thermo Fisher Scientific (1 mentions)
Cd3 fitc, by BD (1 mentions)
Hla dr fitc, by BD (1 mentions)
Cd19 apc, by BD (1 mentions)
Igd pe, by BD (1 mentions)
Cd4 percp, by BioLegend (1 mentions)
Cd38 pe cy7, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions)
Glomax explorer microplate reader, by Promega (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Rnase a, by Merck Group (1 mentions)
F4 80 percp cy 5, by BD (1 mentions)
γδt pe, by BD (1 mentions)
Perm wash reagent, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Cd4 apc, by BD (1 mentions)
7 protocols using flow jo software version 7
1
Phenotyping of PBMC Surface Markers
2
Multiparameter Flow Cytometry Analysis
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Expression of the following markers on CD4 T cells and CD19 B cells was assessed after 4 days in culture using the following monoclonal antibodies (mAb): CD4-PerCP (Biolegend, San Diego, CA, USA), CD19-APC (BD, Erembodegem-Aalst, Belgium), CD25-PE (DAKO, Glostrup, Denmark), and HLA-DR-FITC (BD). For analysis of B cell differentiation the following monoclonal antibodies were used: CD3-FITC (BD Pharmingen, San Diego, CA, USA), CD19-APC-Alexa Fluor750 (BD), CD20-eFluor450 (eBioscience, San Diego, CA, USA) CD27-APC (ImmunoTools GmbH, Friesoythe, Germany), CD38-PE-CY7 (BD), and IgD-PE (BD). IgG1-FITC/IgG1-PE (Immunotech, Marseille, France) was used as isotype control.
Ki67 intracellular staining was performed using mouse-anti-human Ki67-FITC staining set (eBioscience) following manufactures instructions. Cell acquisition was done using a FACScan flow cytometer and data were analysed with FlowJo software, version 7.5 (Tree Star Inc., Oregon, USA).
Ki67 intracellular staining was performed using mouse-anti-human Ki67-FITC staining set (eBioscience) following manufactures instructions. Cell acquisition was done using a FACScan flow cytometer and data were analysed with FlowJo software, version 7.5 (Tree Star Inc., Oregon, USA).
3
Quantifying Chemokine Receptor Expression on B Cells
4
Cell Viability and Cell Death Assay
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Cell viability was measured by CellTiter-Glo luminescent cell viability assay (Promega # G7572) using a Glomax Explorer Microplate Reader (Promega), according to the manufacturer’s protocols. For the propidium iodide (PI) staining, cancer cells were collected, washed with phosphate-buffered saline (PBS) and resuspended in PBS containing 1% FBS, 50 µg/ml PI (Sigma-Aldrich #P4170) and 10 µg/ml RNase A (Omega bio-tek #AC118). Flow cytometry analysis was performed using Becton Dickinson DXP flow cytometer and the data were analyzed by FlowJo software version 7.5 (Tree Star Inc., Ashland, OR, USA).
5
Cell Cycle Analysis by Flow Cytometry
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A total of 1×106 cultured cells were seeded in 60-mm culture dishes and transfected with pCS2-MT, pEGFP-N3, pCS2-MT/Axin or pEGFP-N3/APC5 plasmids using Lipofectamine 2000. At 72 h following transfection, cells were washed twice with ice-cold PBS, resuspended in1 ml pre-cooled 70% ethanol and fixed at 4°C for 24 h. Following fixation, cells were resuspended in 0.5 ml PBS. Propidium iodide and RNaseA (Sigma-Aldrich; Merck Millipore) were added to the cell suspension at a final concentration of 50 µg/ml, and incubated at 37°C for 30 min. Cell cycle distribution was assessed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) by measuring red fluorescence at the excitation wavelength of 488 nm, with Flow Jo software version 7.0 (Tree Star, Inc., Ashland, OR, USA).
6
Lymphocyte Immunophenotyping in Mice
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Each mouse lymph nodes and spleen were minced separately and passed through a 70 μm cell strainer to obtain single-cell suspensions. Single cells from the draining lymph nodes were used for the detection of innate immune cells and were stained with the following monoclonal antibodies: F4/80-PerCP-Cy 5.5, CD11c-PE, CD11b-APC, and Ly6G-FITC (BD Biosciences, USA). The γδT and Th17 cells from the spleen samples were detected with the following fluorescent antibodies: CD45-PerCP-Cy 5.5, γδT-PE, CD4-APC, and IL-17A-PE (BD Biosciences, USA). For the surface antigen staining, cells were incubated with the antibodies for 30 min at room temperature (RT) after washing the cells with PBS. To stain the intracellular cytokines, the cells were stimulated with a cell stimulation cocktail (eBiosicence) for 4 h. After that, the cells were stained for surface antigens and then fixed with BD Cytofix buffer, permeabilized by using Perm/Wash reagent (BD Biosciences) and then stained with anti-IL-17A antibodies. The cells were acquired using an LSR II Flow Cytometer (BD Biosciences), and the data were analyzed using the Flow Jo software version 7.0 (Tree Star, California, USA).
7
Detecting TLR7 in Human Basophils
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To detect expression of TLR7 in human blood basophil cells, blood cells were challenged with or without ASWE,HDME or PPAE (all at a concentration of 1.0 μg/ml) for 60 min at 37℃, respectively, and 2 μg/ml brefeldin A was also added into the tube.Cells were then incubated with human Fc receptor blocking solution and a live/dead cell dye (Zombie Green TM Fixable Viability kit for 15 min, and each labelled monoclonal antibody including APC/Cy7-conjugated mouse anti-human CCR3, PE/Cy7-conjugated mouse anti-human CD123, PerCP/Cy5.5-conjugated mouse anti-human HLA-DR was added into the tube.
After red blood cells being lysed, resuspended leucocytes were xed and permeabilized using Cyto x/Cytoperm TM Fixation/Permeabilization kit according to the manufacturer's instructions.This was followed by adding a PE-conjugated anti-human TLR7 antibody into the tube and incubated at 4℃ for 30 min.
Finally, cells were resuspended in uorescence activated cell sorting (FACS)-ow solution and analysed with FACS Verse ow cytometer (BD Biosciences, San Jose, CA). A total of 10,000 events in live cell gate were analysed for each sample. Data were analysed with FlowJo software version 7.0 (Treestar, Ashland, OR, USA). Dead cells and doublets were excluded from analysis by live/dead cell dyes.
After red blood cells being lysed, resuspended leucocytes were xed and permeabilized using Cyto x/Cytoperm TM Fixation/Permeabilization kit according to the manufacturer's instructions.This was followed by adding a PE-conjugated anti-human TLR7 antibody into the tube and incubated at 4℃ for 30 min.
Finally, cells were resuspended in uorescence activated cell sorting (FACS)-ow solution and analysed with FACS Verse ow cytometer (BD Biosciences, San Jose, CA). A total of 10,000 events in live cell gate were analysed for each sample. Data were analysed with FlowJo software version 7.0 (Treestar, Ashland, OR, USA). Dead cells and doublets were excluded from analysis by live/dead cell dyes.
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