Dissected adipose tissue was cut into small pieces and suspended in RPMI (Sigma, USA) with 20% heat-inactivated FCS (Lonza, Switzerland) before homogenization for 1 hour in a shaker-incubator at 37 °C with 2 mg/mL collagenase II (C6885, Sigma, USA). Homogenized adipose tissue was processed as previously described55 (link) using FACS buffer (PBS with 1% heat-inactivated FCS and 0.1% NaN3) without fixation and permeabilisation. Cells were recorded on a LSRII (BD Biosciences, USA) flow cytometer, and data further analysed using Flowjo software (V10.0.7, Treestar). Gating strategies are shown in Fig. S6 . The following antibodies for surface staining were used: CD45/PerCP (BioLegend, 30-F11), Siglec-F/PE (BD, E50-2440), CD11b/V500 (BD, M1/70), F4/80/APC (BioLegend, BM8), CD11c/APC-Cy7 (BD, HL3), CD45/AF647 (BD, 30-F11), CD4/AF488 (eBioscience, GK1.5), CD8a/FITC (BD, 53–6.7), CD11b/FITC (eBioscience, M1/70), CD49b/FITC (eBioscience, HMa2), F4/80/FITC (eBioscience, BM8), NK1.1/FITC (BD, PK136), FcεR1/PerCP-eF710 (eBioscience, MAR-1), CD19/PerCP-Cy5.5 (eBioscience, 1D3), CD11c/PE-cy7 (BD, HL3), ST2-biotin (MD biosciences, 101001B), Streptavidin-PE (eBioscience).
Cd11b v500
Manufactured by BD
Sourced in United States
CD11b-V500 is a fluorescent-labeled antibody used in flow cytometry to identify and study cells expressing the CD11b antigen. It provides a specific marker for the detection and analysis of myeloid cells, such as monocytes, macrophages, and granulocytes.
Automatically generated - may contain errors
Lab products found in correlation
Software, by FlowJo (3 mentions)
Cd11c apc cy7, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
F4 80 apc, by BioLegend (2 mentions)
Siglecf pe, by BD (1 mentions)
Cd11c pe cy7, by BD (1 mentions)
Streptavidin pe, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Lonza (1 mentions)
Cd45 af647, by BD (1 mentions)
Nk1.1 fitc, by BD (1 mentions)
Cd19 percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd11b fitc, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Merck Group (1 mentions)
Cd45 percp, by BioLegend (1 mentions)
F4 80 fitc, by Thermo Fisher Scientific (1 mentions)
Collagenase 2, by Merck Group (1 mentions)
Cd8a fitc, by BD (1 mentions)
Cd86 pe cy7, by BD (1 mentions)
Cd4 v500, by BD (1 mentions)
Analysis software, by FlowJo (1 mentions)
8 protocols using cd11b v500
1
Flow Cytometric Profiling of Adipose Tissue
2
Multiparameter Flow Cytometry of Immune Cells
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Prior to flow cytometry, cells were washed in staining buffer (0.05% (w/v) BSA, 2 mM EDTA in 1× PBS) and treated with FcR blocking reagent (Miltenyi Biotec) for 10 min. Subsequently, In vitro differentiated single cell clones and Dox-pDC were stained with the following antibodies for 30 min at 4°C: CD11c-APC, MHC-I-FITC, MHC-II-PE, SiglecH-PE, CD86-PE-Cy7, CD289 (TLR9)-FITC, CD11b-V500, B220-PerCP, CD8α-APC-Cy7 (all BD Biosciences) and CD9-FITC (Thermo Fisher). T lymphocytes were stained with the following antibodies: CD3-FITC, CD4-V500, CD8α-APC-Cy7, CD44-APC and IFNγ-APC-Cy7 (all BD Biosciences); CD62L-PerCP-Cy5.5 and RORγt-PerCP-ef710 (all Thermo Fisher Scientific). Flow cytometry was performed using LSRII and FlowJo analysis software (V10; FlowJo, Ashland, USA).
3
Flow Cytometric Characterization of Mouse and Human Immune Cells
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Flow cytometric analyses were conducted using an LSR II flow cytometer (BD Biosciences). Data were evaluated using FlowJo software (Version 7.6.5; Flowjo). All antibodies against human or mouse cells were used at appropriate dilutions, as determined by previous titration. Doublet discrimination was carried out and non-viable cells were excluded by 4,6 diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich). Mouse cells were characterized using the antibodies as follows: CD3-V500, CD4-V450, CD11c-APC Cy7, SiglecF-AF647, Ly6G-FITC, CD11b-V500, B7-1-V450, B7-2-PE Cy7, CD62L-FITC (BD Biosciences); mPDCA1-APC, B220-PE (Miltenyi Biotec); CD8-PE Cy7, F4/80-PerCP, SiglecH-PE, TLR4-AF488, TLR9-FITC, MHC-I-FITC, MHC-II-V450, PD-L1-PerCP, ICOS-L-PE, OX40L-APC (eBioscience); TLR7-PE (Abcam); p75NTR-AF488 (Advanced Targeting Systems), CD45-Pacific Blue (Biolegend), and panTrk-FITC (Cell Signaling Technology). Human cells were characterized using the antibodies as follows: TrkA-PE (R&D Systems); BDCA-2-FITC, BDCA-4-PE, p75NTR-APC, p75NTR-FITC, p75NTR-PE (Miltenyi Biotec); CD45-V500, CD3-PE, CD4-FITC, CD8-PerCP, FcεRIα-FITC, IL-3R-PE Cy7, CD184-PE Cy7, MHC-I-PE Cy7, MHC-II-PE, CD80-V450, CD86-PE, CD83-V500, OX40L-V500, PD-L1-PE Cy7, CCR7-V450, CCR9-APC (BD Biosciences), CD3-APC eFluor780, CD4-APC, CD8-PE Cy7, CD25-PE (eBioscience), and CD69-PerCP Cy5.5 (BioLegend).
4
Cabozantinib Modulates Immune Populations
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To analyze immune cell populations in the presence or absence of cabozantinib, CEA-Tg C57BL/6 mice (n =5/group) were fed control or cabozantinib-containing diet for 10 or 35 days. Spleens were harvested and adjusted to a single-cell suspension. Red blood cells were removed using ACK Lysing Buffer (Quality Biologicals Inc., Gaithersburg, MD). Remaining splenocytes were blocked with mouse Fc Block (BD Biosciences) for 30 min at 4°C, then stained with the following antibodies: CD3e-V500, CD4-AF700, CD8a-Pacific Blue, CD25-FITC, CD11b-V500, Gr-1-AF700, CD49b-FITC, CD19-PE-Cy7, CD11c-PerCP-Cy5.5 (BD Biosciences) and MHC II-APC (eBioscience) for 60 min at 4°C. For intracellular staining of cells with FoxP3-PE, cells were incubated with Fixation/Permeabilization solution for 16 h at 4°C, then incubated with the antibody in Permeabilization Buffer (eBioscience) for 60 min at room temperature. All samples were acquired on an LSR II flow cytometer and analyzed using FlowJo software.
5
Isolation and Characterization of Colon Tumor Immune Cells
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Isolation of mononuclear cells from colon tumor tissue was performed as described previously (28 (link)). Single cell suspension was washed twice in PBS with 0.5% BSA and 2 mM EDTA and stained with antibodies for CD45-PE-Cy7, CD11b-V500, CD11c-PerCP, IL-12p35-PE, and IL-23p19-APC (BD Biosciences). Flow cytometric analyses were conducted using an LSR II flow cytometer (BD Biosciences). Data were evaluated using FlowJo software (Version 7.6.5; FlowJo).
6
Brain Immune Cell Profiling in Tumor-Bearing Mice
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On day 28 post tumor cell inoculation, mice were terminated and the brains of the treatment groups and one tumor-free (normal) littermate were dissected and immediately transferred in ice-cold HBSS. Brains were digested in a buffer solution containing HBSS, Collagenase P (0.2 mg/mL), Dispase II (0.8 mg/mL), DNase I (0.01 mg/mL), and Collagenase A (0.3 mg/mL) for 60 min at 37°C under gentle rocking as described before.41 (link) Briefly, the digested cells were pre-incubated with rat anti-mouse FccIII/II receptor (CD16/CD32) blocking antibody for 5 min at 4°C, and then stained with the fluorochrome-conjugated antibodies (1:100). PI (1:3,000; Thermo Fisher Scientific) was added for live gating. Flow cytometry was performed using FACSDiva software on a LSRII (BD Biosciences; San Jose, CA, USA) and analyzed using FlowJo software (Tree Star; Ashland, OR, USA).
The cells were stained with anti-mouse antibody cocktail: CD45 AF700 (clone 30-F11, eBioscience), Gr-1 PerCP Cy5.5 (clone RB6-8C5, BioLegend), CD11b V500 (clone M1/70, BD), CD11c PE Cy7 (clone N418, BioLegend), F4/80 APC Cy7 (clone BM8, BioLegend), CD206 BV421 (clone C068C2, BioLegend), MHC class II PE-Cy5 (clone M5/114.15.2, eBioscience), T cell receptor (TCR)-β FITC (clone H57-597, BioLegend), and NK1.1 APC (clone PK136, BioLegend).
The cells were stained with anti-mouse antibody cocktail: CD45 AF700 (clone 30-F11, eBioscience), Gr-1 PerCP Cy5.5 (clone RB6-8C5, BioLegend), CD11b V500 (clone M1/70, BD), CD11c PE Cy7 (clone N418, BioLegend), F4/80 APC Cy7 (clone BM8, BioLegend), CD206 BV421 (clone C068C2, BioLegend), MHC class II PE-Cy5 (clone M5/114.15.2, eBioscience), T cell receptor (TCR)-β FITC (clone H57-597, BioLegend), and NK1.1 APC (clone PK136, BioLegend).
7
Multicolor Flow Cytometry for Liver Immune Cells
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Cells were labeled with fluorescence tagged antibodies; using anti-mouse CD16/CD32 (mouse Fc blocker, Clone 2.4G2) (BD Pharmingen) and the Live/Dead fixable aqua dead cell stain kit with detection at 405 nm (Thermo Fisher Scientific). Infiltrating macrophages (CD11b+, F4/80low) and Kupffer cells (CD11b+, F4/80high) were gated using eFlour 450-conjugated anti-mouse CD45 (Clone 30-F11), PE-F4/80 (Clone BM8) (eBioscience), anti-mouse APC, APC-Cy7, or V500-CD11b (Clone M1/70) (BD Pharmingen), as well as with anti-mouse FITC, PerCP-Cy5.5 or APC-Cy7-Ly-6C (Clone AL-21) and anti-mouse APC or PE-CCR2 (Clone #475301) (R&D Systems). LSECs (CD11b−, CD146+) were also analyzed using eFlour 450-conjugated anti-mouse CD45 (Clone 30-F11) and anti-mouse PE-CD146 (Clone ME-9F1) (BD Pharmingen). Cells were read with FACS LSRII (BD Biosciences), and data were analyzed with FlowJo software (FlowJo LLC).
8
Staphylococcus aureus Infection in Mice
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Eight- to 9-week-old C57BL/6 mice were injected retro-orbitally with PBS or approximately 1 × 107 CFU of S. aureus. One day after infection, mice were euthanized. The livers and kidneys were removed, homogenized in RPMI, and digested in 200 U of type VIII collagenase (Sigma-Aldrich, C2139) and 0.2 mg of DNase I (Sigma-Aldrich, DN25). Homogenate was strained, washed, and loaded onto a 40/80% Percoll gradient. The gradient was centrifuged, and the leukocyte layer was removed. RBCs were lysed with 1× BD Pharm Lyse and washed. Cells were stained with V500 CD11b (BD, 562128), PE-Cy7 CD45 (BioLegend, 147704), PE Ly6G (BD, 551461), APC F4/80 (BioLegend, 123116), and Fc block at 1:200 dilution in PBS for 20 min on ice, washed twice, and resuspended in FACS fixing buffer [PBS + 2% FBS + 2% PFA + 0.05% (w/v) sodium azide]. Flow cytometry data were acquired using a CytoFLEX flow cytometer (Beckman Coulter), and data were analyzed using FlowJo software (TreeStar Inc.).
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