After removing erythrocytes, SVF was resuspended in PBS and incubated with FcR Blocking Reagent (130-092-575, Miltenyi Biotec) in the dark on ice for 30 min. Then cells were stained with antibodies of PE-CY7-conjugated anti-F4/80 (25-4801-82, eBioscience), PE-conjugated anti-CD11c (557401, BD) and APC-conjugated anti-CD206 (141708, Biolegend). FCM was performed by using LSRFortessa (BD).
Pe conjugated anti cd11c
Manufactured by BD
Sourced in United States
PE-conjugated anti-CD11c is a fluorescently labeled antibody that binds to the CD11c cell surface antigen. CD11c is a marker expressed on dendritic cells and certain other myeloid cells. This reagent can be used in flow cytometry applications to identify and study these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Fitc conjugated anti cd86, by BD (3 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Lsrfortessa, by BD (1 mentions)
Apc conjugated anti cd206, by BioLegend (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
D glucose, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd11c, by BD (1 mentions)
Pe conjugated anti ccr1, by R&D Systems (1 mentions)
Pe conjugated anti ccr2, by R&D Systems (1 mentions)
D luciferin, by Promega (1 mentions)
Facscanto 2, by BD (1 mentions)
Ova alexa488, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Apc conjugated anti cd103, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
12 protocols using pe conjugated anti cd11c
1
Immunophenotyping of Adipose Stromal Cells
2
Murine Dendritic Cell Differentiation
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RPMI 1640 was purchased from Life Technologies Corporation (Grand Island, NY, USA), containing 4.5 g/L D-Glucose, L-Glutamine and 110 mg/L Sodium Pyruvate. GM-CSF, IL-4 and TNF-α were purchased from Peprotech Asia (Revohot, Israel). Lipopolysaccharide (LPS) was purchased from Sigma Aldrich (Steinheim, Sweden). D-luciferin was purchased from Promega Corporation (Madison, WI, USA). The following antibodies were used for FACS analysis: fluorescein isothiocyanate (FITC)-conjugated anti-CD86, FITC-conjugated anti-I-A[b], FITC-conjugated anti-H-2Kb, Phycoerythrin (PE)-conjugated anti-CCR7, PE-conjugated anti-mouse CD40, PE-conjugated anti-CD11c and FITC conjugated anti-CD11c were purchased from BD PharMingen (SanDiego, CA). PE-conjugated anti-H-2Kb (SIINFEKL) was purchased from BioLegend (San Diego, CA). PE-conjugated anti-CCR1, PE-conjugated anti-CCR2, PE-conjugated anti-CXCR1 and PE-conjugated anti-CCR7 were purchased from R&D Systems (Minneapolis, MN, USA). All the isotype control antibodies were purchased from the same company as the detecting antibodies.
3
Quantifying Antigen Uptake by Bone Marrow-Derived Dendritic Cells
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Antigen uptake by BMDCs was measured using the standard method. OVA-Alexa488 (Invitrogen) was used as the model antigen. BMDCs (2×105) were incubated for various time points at 37 °C with OVA-Alexa488 (50 µg/mL) or OVA-Alexa488-MNPs (OVA-Alexa488 50 µg/mL/MNPs 25, and 50 µg/mL) particles. Uptake was terminated by washing the cells with ice-cold fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline containing 1% FBS and 0.1% NaN3). Cells were stained with PE-conjugated anti-CD11c (BD Biosciences). Alexa488 fluorescence of CD11cpositive cells was measured using an FACS CantoII (BD Biosciences). The morphology of the BMDCs was examined using a Confocal Laser Scanning Microscope (CLSM) (PE: excitation 496, emission; 578 FITC: excitation 488, emission 519).
4
Intestinal DC Uptake of FITC-IK Nanoparticles
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For easier detection of cell uptake by intestinal DCs, naïve mice were orally administered with 1mg of FITC-IK and FITC-IK NPs, respectively. After 6 h, small intestine was removed and flow cytometry analysis of the intestinal lamina propria mononuclear cells (LPMCs) was performed. The cell suspensions were stained with PE-conjugated anti-CD11c and APC-conjugated anti-CD103 (BD Biosciences Pharmingen). Isotype IgG was used as control antibodies. CD11c+CD103+ cells were gated for intestinal DCs and then FITC intensity was directly analyzed using a FACS Calibur Flow cytometer (BD Biosciences). The data were analyzed using Flowjo software (7.6.1).
For fluorescence analysis of in vivo cell uptake, intestinal cryosections of mice receiving free FITC-IK and FITC-IK NPs were prepared. The sections were incubated with DAPI in order to visualize nuclei. Images of positive staining were taken using a fluorescence microscope (Olympus, Tokyo, Japan).
For fluorescence analysis of in vivo cell uptake, intestinal cryosections of mice receiving free FITC-IK and FITC-IK NPs were prepared. The sections were incubated with DAPI in order to visualize nuclei. Images of positive staining were taken using a fluorescence microscope (Olympus, Tokyo, Japan).
5
Th17 cell polarization by HMGB1-activated macrophages
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CD4+T cells were isolated by magnetic activated cell sorting (MACS) using CD4 antibodies (Miltenyi Biotec, Germany) according to the manufacturer’s instructions. Isolated T cells were cocultured with macrophages treated by 100 ng/ml rHMGB1 (H4652, Sigma Aldrich, USA), BSA or without, respectively for 4 days36 (link). Th17 cell polarization was assessed according previously describe (50 ng/ml PMA and 1 μg/ml ionomycin)37 (link) (both from Sigma Aldrich, USA). GolgiPlug (BD Pharmingen, USA) was added during the last 5 h. Cells were fixed and permeabilized and stained with FITC-conjugated anti-IL-17 (BD Pharmingen, USA).
Macrophages were surface-stained with PE-conjugated anti-CD11c and FITC-conjugated anti-F4/80 antibody. After washing with phosphate buffered saline (PBS), stained cells were resuspended and analyzed.
All the samples were analyzed by FACS Calibur (BD Biosciences, USA).
Macrophages were surface-stained with PE-conjugated anti-CD11c and FITC-conjugated anti-F4/80 antibody. After washing with phosphate buffered saline (PBS), stained cells were resuspended and analyzed.
All the samples were analyzed by FACS Calibur (BD Biosciences, USA).
6
Phenotypic Analysis of Dendritic Cells
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Phenotypic analysis was performed by direct immunofluorescence staining of DC cell surface markers. Cells (in PBS) were stained with the following antibodies at 4 °C for 20 min: Phycoerythrin (PE)-conjugated anti-CD11c (Clone N418); Fluorescein isothiocyanate (FITC)-conjugated anti-CD14 (Clone Sa14-2); PE-conjugated anti-CD40 (Clone 3/23); FITC-conjugated anti-CD54 (Clone 3E2); PE-conjugated anti-CD80 (Clone 16-10A1); FITC-conjugated anti-CD86 (Clone PO3); FITC-conjugated anti-MHC I (Clone KH95), or PE-conjugated anti-MHC II (Clone 2G9) (all antibodies were purchased from BD Pharmingen, San Diego, CA, USA), plus appropriate isotype controls. Cells were then analyzed using a 3-color FACSCalibur cytometer (BD Biosciences, Mountain View, CA). Data were collected from 10,000 events and analyzed using FlowJo10 software (BD Biosciences).
7
Phenotyping Peritoneal Immune Cells
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6-wk-old BALB/cJRj mice (Charles River) were i.p. infected with 105 parasites and sacrificed on days 4 and 7 p.i. Immediately after being killed, mice were peritoneally lavaged with PBS, and recovered lavage fluid was centrifuged at 500 g for 8 min. Peritoneal cells were washed in stain buffer (PBS containing 2% FBS and 0.1 mM EDTA), and cells were then pretreated at 4°C for 1 h with mAb 2.4G2 to block nonspecific binding to Fcγ receptors. Thereafter, cells were incubated for 1 h with fluorescently conjugated antibodies for cell surface markers from BD: PE-conjugated anti-CD11b, PE-conjugated anti-Gr1, PE-conjugated anti-CD11c, and APC-conjugated anti–Ly-6G. Isotype controls consisted of PE-conjugated rat IgG2b, PE-conjugated hamster IgG1, and APC-conjugated rat IgG2a, provided by BD. Analysis of stained cells was performed with a FACSCalibur flow cytometer (BD). For all samples, 100,000 cells were analyzed for plot generation.
8
Identification of Fusion Cells
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Fusion cells were identified by the morphology, fluorenscence microscope and flow cytometry. The morphology was carried out by inverted microscope; tumor cells were pre-labeled with fluorescein isothiocyanate (FITC)-conjugated anti-Muc1 (BD CO., USA). The hybrid cells were incubated with phycoerythrin (PE)-conjugated anti-CD11c (BD) for 30 min at 4°C. Cells were washed, fixed and analyzed by fluorenscence microscope and FACScan (BD) with CellQuest analysis software.
9
Tracking Nanoparticle Biodistribution in Mice
10
Flow Cytometry Analysis of Immune Cells
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LPMCs suspended in staining buffer (PBS containing 1 % FBS, 5 mM EDTA and 0.02 % NaN3) were incubated with anti-CD16/32 monoclonal antibodies (mAb) for 30 min at 4 °C to block nonspecific binding. Then, cells were stained for 30 min at 4 °C with a combination of the following fluorescent mAbs: FITC-conjugated anti-Ly6G or anti-DX5 (BD Biosciences), PE-conjugated anti-CD11c (BD Biosciences), PE-Cy7-conjugated anti-CD11b (BD Biosciences), Alexa Fluor 647-conjugated anti-Ly6C (BioLegend), APC-conjugated anti-CD3ε (eBioscience) or anti-CD19 (eBioscience). Dead cells were gated out by staining with 7-aminoactinomycin D (7-AAD) (Life Technologies, Carlsbad, CA). Stained cells were acquired using FACSCalibur (BD Biosciences) or FACSAriaIII (BD Biosciences), and data were analyzed with FlowJo software (TreeStar Inc., Ashland, OR).
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