Tumor-infiltrating and splenic lymphocytes were analyzed by flow cytometry. In brief, tumor and spleen tissues were harvested and digested with collagenase A and hyaluronidase at 37 °C for 40 min. After lysis of the red blood cells (RBCs), the dissociated cells were dispersed with 1 mL of HBSS. For intracellular cytokine staining, the cells from the tumor and spleen tissue were permeabilized with 0.1% triton X-100 for 15 min. The density of total cells was 5×106/mL. The tumor infiltrating immune cells were stained with the following fluorescence-labeled antibodies: FITC-conjugated anti-mouse CD8a, FITC-conjugated anti-mouse CD4, PE-conjugated anti-mouse Foxp3, FITC-conjugated anti-mouse CD11b, and PE-conjugated anti-mouse Gr-1 (BD, New South Wales, Australia). Flow cytometry was performed in quintuplicate for each group. Analysis was performed on a FACSCalibur flow cytometer and analyzed using Cell Quest software (BD Biosciences, San Jose, CA).
Fitc conjugated anti mouse cd11b
Manufactured by BD
Sourced in United States, Australia
The FITC-conjugated anti-mouse CD11b is a laboratory reagent used to detect the expression of the CD11b antigen on mouse cells. CD11b is a cell surface integrin molecule that is expressed on myeloid cells, including monocytes, macrophages, and granulocytes. The FITC fluorescent label allows for the visualization and quantification of CD11b-positive cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Fitc conjugated anti mouse cd4, by BD (4 mentions)
Cellquest software, by BD (4 mentions)
Pe conjugated anti mouse foxp3, by BD (3 mentions)
Pe conjugated anti mouse gr 1, by BD (2 mentions)
Fitc conjugated anti mouse cd8a, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Apc conjugated anti mouse gr 1, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Mouse regulatory t cell staining kit, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti mouse cd11c, by BD (1 mentions)
Cell stimulation cocktail, by BD (1 mentions)
Facscalibur system, by BD (1 mentions)
Pe conjugated anti mouse il 17a, by BD (1 mentions)
Facs caliber flow cytometer, by BD (1 mentions)
Balb c mice, by Charles River Laboratories (1 mentions)
Anti human cd3 fitc, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 700 conjugated anti mouse cd45, by BioLegend (1 mentions)
Pecy7 conjugated anti mouse nk1, by BD (1 mentions)
Kaluza flow analysis software, by Beckman Coulter (1 mentions)
Pe conjugated anti mouse f4 80, by BD (1 mentions)
9 protocols using fitc conjugated anti mouse cd11b
1
Tumor Immune Cell Profiling by Flow Cytometry
2
Tumor and Spleen Immune Profiling
3
Multiparametric Analysis of Murine Immune Cells
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Mice were euthanized on the 126th day. Spleens, inferior epigastric lymph nodes and mesenteric lymph nodes were collected and single-cell suspensions were then obtained from mononuclear cells (MNCs). Cells were stained with fluorescent surface antibodies: FITC-conjugated Anti-Mouse CD4, APC-conjugated Anti-mouse CD8a, FITC-conjugated Anti-mouse CD11b, FITC-conjugated Anti-mouse CD11c, APC-conjugated Anti-mouse Gr-1, APC-conjugated Anti-mouse MHCII (all from BD Biosciences, San Jose, CA, USA). For staining of cytokines: APC-conjugated Anti-mouse IFNγ, PE-conjugated Anti-Mouse IL-4 and PE-conjugated Anti-Mouse IL-17A were purchased from BD Biosciences. Mouse regulatory T cell staining kit (eBioscience, San Diego, CA, USA) was used according to the manufacturer’s instructions. For intracellular cytokine staining, cells were restimulated with Cell Stimulation Cocktail (BD Biosciences) for 4 h at 37 °C. Cell phenotyping and sorting were performed on a BD FACS Calibur system (BD Biosciences) and data were analyzed using FlowJo software.
4
BALB/c Mice Immune Cell Analysis
5
Flow Cytometry Immunophenotyping Protocol
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For flow cytometry analysis, cell surface staining was performed using PE-conjugated anti-IFNAR1 (SinoBiological, 50469-R110-P), PE-conjugated anti-IFNAR2 (R&D Systems, ABVR0220021), PerCP-Cy5.5-conjugated anti-mouse Ly6G (BD Bioscience, 560602), FITC-conjugated anti-mouse CD11b (BD Bioscience, 553310), and Alexa Fluor 700-conjugated anti-mouse CD45 (BioLegend, 103128). Cell staining was performed at 4 °C for 30 min and acquired with BD FACS Canto II. FACS data were analyzed using a FlowJo software (FlowJo). For flow sorting, PBMCs were collected with cold 1× PBS and incubated for 30 min at 4 °C with either the FITC anti-human CD3 (eBioscience, 4312299) (T cells), the Alexa-Flour 488 anti-human CD19 (Biolegend, 302219) (B cells), or the PerCP-Cy5.5 anti-human CD14 (BD Bioscience, 550708). After washing, the cells were subjected to sorting using the BD FACS Canto II.
6
Intracellular PTX3 and Immune Cell Analysis
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To analyze PTX3 intracellular expression, transfected HEK 293T cells were fixed with 2% PFA and permeabilized with 0.1% Saponin (Sigma Aldrich) in PBS. Indirect intracellular staining was performed with rat anti-PTX3 (MNB4, Abcam) primary antibody, followed by AF488-conjugated anti-rat (Life Technologies) secondary antibody. To identify the various cell populations present in splenocytes, peritoneal lavage and quadriceps harvested from mice, cells were first incubated with anti-mouse CD16 / CD32 (FC block, BD Pharmingen) and stained with the following antibodies: APC-conjugated anti-mouse GR1, PE-conjugated anti-mouse F4/80, FITC-conjugated anti-mouse CD11b, APC-conjugated anti-mouse Ly6c, APC-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD19, PE-conjugated anti-mouse CD45, or PE-Cy7-conjugated anti-mouse NK1.1 (BD Pharmingen). For detection of alphavirus antigens, indirect intracellular staining was performed using mouse monoclonal anti-alphavirus (3581, Santa Cruz) primary antibody, followed by AF488-conjugated anti-mouse (Life Technologies) secondary antibody. Data acquisition was performed using CyanADP (Beckman Coulter), and analysis was done by Kaluza Flow Analysis Software (Beckman Coulter).
7
Immune Cell Phenotyping in Tumor Microenvironment
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In order to evaluate the phenotype of different immune cell populations, cells derived from the spleen or tumor of vehicle- or AZD1480-treated mice were stained with the following antibodies: phycoerythrin (PE)-Cy7-conjugated anti-mouse CD3 (BioLegend), Alexa Fluor 700 (AF700)-conjugated anti-mouse CD4 (BD Biosciences), AF647-conjugated anti-mouse CD8 (BioLegend), Horizon V450-conjugated anti-mouse CD45 (BD Biosciences), peridinin chlorophyll protein (PerCP)-Cy5.5-conjugated anti-mouse CD4 (BD Biosciences), PE-conjugated anti-mouse CD25 (eBioscience), AF700-conjugated anti-mouse CD127 (eBioscience), AF647-conjugated anti-mouse CD11c (BioLegend), PE-conjugated anti-mouse CD11b (BD Biosciences), fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD86 (BD Biosciences), allophycocyanin (APC)-H7-conjugated anti-mouse CD80 (BD Biosciences), FITC-conjugated anti-mouse CD11b (BD Biosciences), AF647-conjugated anti-mouse Ly6G (BioLegend) and PECy7-conjugated anti-mouse Ly6C (BioLegend).
8
Immunostaining and Flow Cytometry Protocols
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Anti-CTLA-4 (9D9) and mouse immunoglobulin G2b (IgG2b) isotype control used in vivo were purchased from BioXCell (West Lebanon, NH, USA). Primary antibodies used for western blot analysis and immunofluorescence staining included anti-STAT3 monoclonal antibody, anti-phospho-STAT3 monoclonal antibody, anti-α-SMA monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), anti-mouse CD8α polyclonal antibody, anti-CD31 polyclonal antibody (Servicebio, Wuhan, China) and anti-GAPDH monoclonal antibody (Zsbio, Beijing, China). Secondary antibody used for western blot analysis and immunofluorescence staining included goat anti-mouse IgG-horseradish peroxidase, goat anti-rabbit IgG-horseradish peroxidase (Abmart, Shanghai, China) and Alexa Fluor 647 conjugated anti-rabbit (Cell Signaling Technology). FITC-conjugated anti-mouse CD8α, PE-conjugated anti-mouse FOXP3, PE-conjugated anti-mouse Gr, and FITC-conjugated anti-mouse CD4 were obtained from Thermo Fisher Scientific (Waltham, MA, USA). FITC-conjugated anti-mouse CD11b, PE-conjugated anti-mouse CD3 and Fc block were purchased from BD Biosciences (San Diego, CA, USA). These primary antibodies were used for flow cytometry assay.
9
Flow Cytometry Characterization of APCs
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In the present study, phycoerythrin (PE)-conjugated rat anti-mouse CD11c, fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse B220, FITCconjugated anti-mouse CD11b and allophycocyanin-conjugated rat anti-mouse major histocompatibility complex (MHC II) (IAbdq, I-Edk), all purchased from Pharmingen (San Diego, CA, USA), constituted the antibodies used for the purity verification of the APCs. The depletion of CD4 + and of CD8 + T-cells was verified with a FITC-conjugated rat anti-mouse CD4 and rat anti-mouse CD8 (YTS169) respectively, detected using a FITC-conjugated AffiniPure F(ab')2 fragment of mouse antirat IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The negative selection of CD4 + T-cells was verified using PE-conjugated rat anti-mouse CD4 and allophycocyanin-conjugated rat anti-mouse CD45 (eBioscience, San Diego, CA, USA). Stained cells were analyzed using either FACSCalibur or FACS Canto II (Becton-Dickinson, Mountain View, CA). The data were then analyzed using CELLQuest™ software (version 3.3; BD Biosciences, San Jose, CA, USA) or with FlowJo software (version 6.3.4; Tree Star Inc., Ashland, OR, USA). Fluorescence data were acquired using logarithmic amplification and the reported fluorescence intensity units represent conversion of channel values according to the logarithmic scale.
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