Bone marrow‐derived DCs were isolated from the bone marrow of 8‐week‐old balb/c mice. Immature DCs (5 × 105 cells/well) plated in a 12‐well plate were treated with TKNP, free R848, and anti‐PD‐L1‐R848/TKNP with the equivalent concentration of R848 (30 μmol/L) for 24 h. Cells were washed with PBS, harvested, and stained with PE‐CD11c, FITC‐CD86, and PerCP‐MHC II (BioLegend) and sorted using FACS.
Cd11c pe
Manufactured by BioLegend
Sourced in United States
CD11c-PE is a fluorescent-labeled antibody that binds to the CD11c antigen, which is expressed on the surface of dendritic cells. It is commonly used in flow cytometry applications to identify and characterize this specific cell population.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 fitc, by BioLegend (4 mentions)
Cd3 apc, by BioLegend (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Cd25 apc, by BioLegend (3 mentions)
Cd4 fitc, by BioLegend (3 mentions)
F4 80 fitc, by BioLegend (3 mentions)
F4 80 pe, by BioLegend (3 mentions)
Cd3 pe cy7, by BioLegend (3 mentions)
Cd11b fitc, by BioLegend (3 mentions)
Collagenase type xi, by Merck Group (2 mentions)
Collagenase type 1, by Merck Group (2 mentions)
Fc blocking antibody, by Thermo Fisher Scientific (2 mentions)
Hyaluronidase type 1 s, by Merck Group (2 mentions)
Cd19 apc cy7, by BioLegend (2 mentions)
Pe cy7 nk1, by BioLegend (2 mentions)
Percy5.5 cd11b, by BioLegend (2 mentions)
Cd86 pe, by BioLegend (2 mentions)
Ly6c fitc, by BioLegend (2 mentions)
Cd4 apc cy7, by BioLegend (2 mentions)
Facsverse, by BD (2 mentions)
32 protocols using cd11c pe
1
Enhancing DC Activation with TKNP and R848
2
Single-cell Isolation and Analysis of Mouse Aorta
3
Lipid-Based Nanoparticle Preparation and Characterization
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The lipids 18: 1TAP (1, 2-dioleoyl-3-trimethylammonium-propane), DOPE (1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine), EPC, 16:0 NBD PE, and 16:0 Liss Rhod PE were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). DSPE-PEG2000-Folic acid was purchased from Nanocs (Boston, MA, USA). Chol (Cholesterol), Collagenase, and DNase I were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The dye DiI was purchased from Beyotime Biotechnology. PTX was purchased from Meilunbio® (Dalian, China). Paclitaxel injection was purchased from HAPHARM Group Co., Ltd. (Harbin, China). The CD9, CD63, and TSG101 antibodies were purchased from Abcam (Cambridge, MA, USA).
The antibodies of flow cytometry (anti-mouse): PE-CD45, FITC-CD4, APC-CD8a, PerCP/Cyanine5.5-CD69, FITC-CD3ε, APC-CD49b (pan-NK cells), APC/Cyanine7-CD335 (NKp46), PE-CD107a (LAMP-1), APC-CD45R/B220, PE/Cyanine7-CD69, FITC-CD45, APC/Cyanine7-F4/80, PE-CD11c, PE/Cyanine7-CD86, APC anti-mouse/human CD11b, PerCP/Cyanine5.5-Ly-6G, FITC-Ly-6C, PE/Cyanine7-CD45, APC-CD25, PE-FOXP3, PE-CD86, and PerCP/Cyanine5.5-CD206 (MMR) were purchased from Biolegend (San Diego, CA, USA).
The dye DiI was purchased from Beyotime Biotechnology. PTX was purchased from Meilunbio® (Dalian, China). Paclitaxel injection was purchased from HAPHARM Group Co., Ltd. (Harbin, China). The CD9, CD63, and TSG101 antibodies were purchased from Abcam (Cambridge, MA, USA).
The antibodies of flow cytometry (anti-mouse): PE-CD45, FITC-CD4, APC-CD8a, PerCP/Cyanine5.5-CD69, FITC-CD3ε, APC-CD49b (pan-NK cells), APC/Cyanine7-CD335 (NKp46), PE-CD107a (LAMP-1), APC-CD45R/B220, PE/Cyanine7-CD69, FITC-CD45, APC/Cyanine7-F4/80, PE-CD11c, PE/Cyanine7-CD86, APC anti-mouse/human CD11b, PerCP/Cyanine5.5-Ly-6G, FITC-Ly-6C, PE/Cyanine7-CD45, APC-CD25, PE-FOXP3, PE-CD86, and PerCP/Cyanine5.5-CD206 (MMR) were purchased from Biolegend (San Diego, CA, USA).
4
Tumor-Infiltrating Lymphocyte Isolation and Characterization
5
Flow Cytometry Technique for Murine Blood
6
Flow Cytometric Characterization of Mouse BMDCs
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For flow cytometry analysis, mouse BMDCs were seeded in a 6-well plate at a density of 0.5×106 cells/well. Treated cells were centrifuged at 300g for 5 min and washed with stain buffer (BD Pharmingen, San Diego, California, USA). Prior to the surface staining, Fc-block was performed using mouse TruStain FcX (BioLegend) for 5 min at room temperature. Subsequently, cells were stained with anti-mouse antibodies (PE-CD11c, FITC-A/I-E, and APC-CD197, BioLegend). Isotype controls were used to confirm antibody specificity. Cells were incubated in the dark for 30 min at 4°C and analyzed using a BD FACSVerse flow cytometer (BD Biosciences, San Diego, California, USA). Dead cells were excluded based on their forward and side scatter or uptake of 7-aminoactinomycin D (Immunostep, Salamanca, Spain). The data were analyzed using FlowJo (BD Biosciences) software.
7
Immune Cell Isolation and Modulation
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Five to seven mouse skin tissues were pooled as one sample for MACS sorting. Mouse skin CD3+ T cells, CD19+ B cells, F4/80+ cells, CD11c+ cells, and Ly6G+ neutrophils were sorted with PE-CD3 (BD Pharmingen, #561824, 145-2C11, 1/100 dilution), PE-CD19 (BD Pharmingen, #561736, 1D3, 1/100 dilution), PE-F4/80 (BioLegend, #123109, BM8, 1/100 dilution), PE-CD11c (BioLegend, #117307, N418, 1/100 dilution), and PE-Ly6G (BioLegend, #127607, 1A8, 1/100 dilution) using anti-PE microbeads (Miltenyi Biotech, #130-048-801) and a MACS system (Miltenyi Biotech, #130-042-201). The purity of sorted cells was >90%. Sorted mouse skin cells were used to extract protein and RNA.
After PBMC preparation, human peripheral monocytes from patients with psoriasis were sorted with PE-CD14 (BD Pharmingen, #555398, M5E2, 1/100 dilution) using anti-PE microbeads and a MACS system. The purity of sorted cells was >90%. Monocytes were treated with KN-93 (10 μM), MEK1/2 (MEK1/2 is upstream of Erk1/2) inhibitor U0126 (20 μM, Sigma, #U120), or p38 inhibitor SB203580 (20 μM, Sigma, #S8307) to extract protein and RNA for 12 h or to test cytokines in the supernatants for 24 h.
After PBMC preparation, human peripheral monocytes from patients with psoriasis were sorted with PE-CD14 (BD Pharmingen, #555398, M5E2, 1/100 dilution) using anti-PE microbeads and a MACS system. The purity of sorted cells was >90%. Monocytes were treated with KN-93 (10 μM), MEK1/2 (MEK1/2 is upstream of Erk1/2) inhibitor U0126 (20 μM, Sigma, #U120), or p38 inhibitor SB203580 (20 μM, Sigma, #S8307) to extract protein and RNA for 12 h or to test cytokines in the supernatants for 24 h.
8
Isolation and Characterization of Adipose Tissue Macrophages
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In each experiment, epididymal and visceral white adipose tissue samples were collected, mixed, and washed with ice-cold phosphate buffer saline (PBS). Adipose tissue samples were minced into small pieces with scissors, suspended in pre-warmed digestion buffer containing 1 mg/mL collagenase II (C6885, Sigma-Aldrich), 1% BSA, and 10 mM HEPES, and incubated at 37 °C for 40 min with vigorous shaking. DMEM containing 2 mM EDTA and 5% FBS was added to stop the digestion. The cell suspension was filtered through a 150-µm nylon filter and centrifuged at 450 × g for 5 min at 4 °C. Adipocytes and supernatant were discarded, and red blood cells were depleted using red blood cell lysis buffer (C3702-500 ml, Beyotime, China). Mouse peripheral blood mononuclear cells were isolated using lymphocyte isolation solution (density: 1.081 g/mL). Finally, cells were blocked with purified anti-CD16/32 antibody (BioLegend, San Diego, CA, USA) and stained with the following antibodies: FITC-CD45.2, APC-F4/80, PerCP/Cy5.5-Ly6C, PE-CD11c, APC/Cy7-Ly6G (BioLegend), and PE-CF594-CD11b (BD Biosciences, USA). Flow cytometry data were acquired using a BD LSRFortessaTM Flow Cytometer and analyzed with FlowJo software (BD Biosciences). For cell sorting, the CD11b+Ly6Chigh monocytes and CD11b+F4/80+ ATMs were sorted with a BD FACSAriaTM III sorter (BD Biosciences).
9
Single-cell Isolation and Analysis of Mouse Aorta
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Single-cell suspensions were prepared from the mouse aortas as described20 (link). Briefly, mice were anesthetized and perfused through the heart with PBS containing 10 U ml−1 heparin to remove blood from all vessels. After careful removal of the perivascular adipose tissue, the aortas were minced into small pieces and digested with 125 U ml−1 collagenase type XI, 60 U ml−1 hyaluronidase type I-s, 60 U ml−1 DNase1, and 450 U ml−1 collagenase type I (all enzymes were obtained from Sigma-Aldrich) in PBS containing 20 mM Hepes at 37°C for 3 hrs. For separating adventitia and media, the aortas were predigested in enzyme cocktail (300U ml−1 Collagenase I and 10U ml−1 Elastase in PBS) at 37°C for 15 minutes and then the adventitia was carefully peeled away from the aorta. After filtering through a 70 μm filter, cells were resuspended in FACS buffer, and incubated with Fc-blocking antibody (eBioscience) for 15 min on ice before being stained with specific antibodies. Antibodies used were as follows: FITC-CD45.2, PE-CD11c, APC-CD3, APC-Cy7-CD19, PE-Cy7-NK1.1, PerCy5.5-CD11b (all antibodies were obtained from Biolegend and used at 1:100 dilution). Cells were also stained with propidium iodide. After washing, immunofluorescence was detected by an LSR II (BD Biosciences). Data were analyzed using FlowJo (Tree Star Inc.) software.
10
Visualization of Adipose Tissue Macrophages
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