The population of each phenotype of immune cells in different groups was evaluated by using flow cytometric analysis as described previously [30 (link)]. In brief, splenocytes were stained with fluorescent antibodies, including anti-CD4-FITC, anti-CD25-PE, anti-Foxp3-PerCP, anti-CD11c-APC, anti-MHCII-FITC, anti-CD86-PE, anti-CD40-PE, anti-CD4-PE (eBiosciences, San Diego, USA), anti-IL-4-APC, anti-CD68-FITC, and anti-CD206-PE (BioLegend, San Diego, USA), according to the manufacturer’s instruction. The FlowJo software was used to analyze the percentages of various phenotypes of immune cells.
Anti cd11c apc
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Anti-CD11c-APC is a fluorochrome-conjugated antibody that binds to the CD11c antigen expressed on dendritic cells and monocytes. It can be used for the identification and enumeration of these cell types in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd86 fitc, by Thermo Fisher Scientific (4 mentions)
Anti cd25 pe, by Thermo Fisher Scientific (3 mentions)
Anti cd86 pe, by Thermo Fisher Scientific (3 mentions)
Anti cd80 fitc, by Thermo Fisher Scientific (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Anti cd4 fitc, by Thermo Fisher Scientific (2 mentions)
Anti cd206 pe, by BioLegend (2 mentions)
Anti mhcii fitc, by Thermo Fisher Scientific (2 mentions)
Anti f4 80 apc, by Thermo Fisher Scientific (2 mentions)
Anti cd11b percp cy5, by BD (2 mentions)
Anti ifn γ apc, by Thermo Fisher Scientific (2 mentions)
Anti mhc 2 pe, by Thermo Fisher Scientific (2 mentions)
Anti cd8 fitc, by Thermo Fisher Scientific (2 mentions)
Anti gr1 fitc, by Thermo Fisher Scientific (2 mentions)
Anti f4 80 pe, by Thermo Fisher Scientific (2 mentions)
Golgiplug, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Anti cd16 32 antibody, by Thermo Fisher Scientific (2 mentions)
Anti foxp3 percp, by Thermo Fisher Scientific (1 mentions)
Anti cd40 pe, by Thermo Fisher Scientific (1 mentions)
26 protocols using anti cd11c apc
1
Flow Cytometric Analysis of Immune Cells
2
Isolation and Analysis of Mouse Liver Non-Parenchymal Cells
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Isolation of mouse liver non-parenchymal cells was as described in this section. Subsequently, antibodies including anti-F4/80-APC (17-4801-82), anti-CD11c-APC (17-0114-82), anti-CD3e-APC (17-0031-82), and anti-CD31-APC (17-0311-82, eBioscience, San Diego, CA) were added to the cell suspension, respectively. After 30 min of incubation at 4°C in the dark, the cells were washed with PBS and incubated with fixation/permeabilization buffer (eBioscience, San Diego, CA). Then, the cells were washed and stained with anti-NLRP3/NALP3-750 (IC7578S, R&D Systems, Eugene, Oregon) for 60 min at 4°C and analyzed with a flow cytometer. FACS was performed on a FACSAria and analyzed with FACSDiva 4.1 (BD Biosciences).
3
Modulation of BMDC Activation by AAT
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Bone marrow-derived dendritic cells (BMDC) were stimulated with IFNγ and IL-1β (5 ng/ml each, Prospec), in the absence or presence of human AAT (0.5 mg/ml). Forty-eight hours later, supernatants were collected for cytokine and nitrite analysis. In the same manner, 24 hours after stimulation, cells were examined by flow cytometry, as described (Lewis et al., 2008b (link)). The following antibodies were used for staining: anti-CD86-FITC, anti-MHC class II-PE and anti-CD11c-APC (all from eBioscience, San Diego, CA).
4
Characterization of CD4+ T Cell Responses in Liver Mononuclear Cells
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Mice were euthanized at indicated time points. Mononuclear cells were purified from the liver and CD4 T cell responses were analyzed as previously described (14 (link)). Briefly, hepatic mononuclear cells were restimulated with bone marrow-derived dendritic cells, pulsed with fixed parasites, and directly incubated at 37°C in the presence of 1/1000 Brefeldin A (GolgiPlug™, BD Biosciences). Cells were then stained with anti-CD4-FITC (BD PharmingenTM, clone GK15), anti-CD3-BV421 (BD Biosciences, clone 14S-2C11), followed by anti-IFN-γ-APC (BD PharmingenTM, clone XMG1.2) after permeabilization with 0.1% saponin. Myeloid cells were stained with anti-CD11b-Pacific Blue (BD HorizonTM, clone MI/70), anti-MHC-II-FITC (BD PharmingenTM, clone 2G9), anti-Ly6C-PerCP (Biolegend, clone HK1.4), anti-Ly6G-PE (Biolegend, clone 1A8), anti-F4/80-PECy7 (Biolegend, clone BM8), and anti-CD11c-APC (eBioscience, clone N418). Flow cytometric analysis was performed with a BD LSRFortessa cell analyzer (Becton Dickinson). Samples were analyzed with Flowjo software.
5
Isolation and Characterization of Immune Cells from Mouse Spinal Cord
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The spinal cord, dissected from mice transcardially perfused with PBS, was minced into 1 mm3 pieces in solution containing 1 mg/mL or 0.1 mg/mL collagenase IV (Worthington Biochemical Corporation, USA) plus 0.4 mg/mL DNase I (Roche, Switzerland), and incubated at 37 °C for 15 min. For isolation of immune cells and microglia, cells were resuspended in 37% Percoll (GE Healthcare, USA) and centrifuged at 780 × g for 20 min. After centrifugation, myelin debris was removed and the cell pellet was collected. Cells were incubated with anti-CD16/CD32 antibodies (eBioscience, USA) for blocking Fc receptors, and then stained with combinations of the following antibodies: anti-CD45-PE-Cy7, anti-CD45-APC-Cy7, anti-CD4-PE, anti-CD8a-FITC, anti-CD8a-APC-Cy7, anti-NK1.1-PErCP-Cy5.5, anti-NK1.1-PE, anti-CD11c-PE, anti-CD11b-APC-Cy7, anti-CD11b-PerCP-Cy5.5 (BD Biosciences, USA), anti-CD4-APC, anti-CD3e-PerCP-Cy5.5, anti-CD86-APC, anti-CD25-PE (eBioscience, USA), anti-CD11c-APC, anti-I-A/I-E (MHC class II)-FITC, anti-CD4-PE-Cy7, anti-CD4-APC, anti-CD68-PE, anti-H-2Kb/H-2Db (MHC class I)-PE, anti-CD206-PE, anti-CD178 (FasL)-PE, and Armenian Hamster IgG Isotype control-PE (BioLegend, USA). Flow cytometry was performed by using FACS Aria and FACS Verse flow cytometers (BD Biosciences, USA) and the data was further analyzed using FlowJo Software (FlowJo, LLC, USA).
6
Multiparametric Flow Cytometry Analysis
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Cells were isolated from BAL, lungs, spleen, and draining lymph nodes and analyzed by flow cytometry as previously described55 (link). Cells were stained with the following antibodies at the concentration of 1:200: CD16/32 (i.e., Fc block, eBioscience), Fixable Viability Dye eFluor 506, anti-Ly6G PE-Cy7 or eFluor 450, anti-CD117 FITC, SiglecF PE, CD11b PE-Texas Red, anti-CD11c APC, anti-MHC II AF700, anti-CD49b PerCP eF710, anti-FcεRIα PECy7, anti-F4/80 APC/Cy7, anti-IL17A PerCP-Cy5.5 (Ebioscience), anti-TCRβ APC-CY7 (Biolegend), anti-CD4 Alexa Fluor 700 or eFluor 450, anti-CD8α PE-Texas Red or PerCP-Cy5.5, anti-TCRδ APC, anti-NK1.1 Allophycocyanin, anti-CD44 V500, anti-CD62L PE-Cy7, anti-CD11b Alexa Fluor 647 (BD Pharmingen), anti-B220 Alexa Fluor 700, PE-PBS57 loaded CD1d tetramer was from the National Institute of Allergy and Infectious Diseases Tetramer Facility. Purified anti-CD3 and CD28 antibodies were from BD Biosciences. In some cases, cells were stimulated with PMA and Ionomycin followed by an analysis of intracellular cytokine as previously described56 (link). Cells were analyzed using a BD FACS Aria II flow cytometer and analyzed with FlowJo software.
7
Fluorochrome-Conjugated Antibodies for Immunostaining
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Fluorochrome-conjugated antibodies used: anti-CD11c-APC, anti-IFNγ-APC, anti-TNFα-PE-cy7, anti-IL-4-PE, anti-IL17-PE, anti-Foxp3-PE (e-Bioscience, Vienna, Austria), and anti-LAMP-1-v450 (BD-Pharmingen). Unconjugated mouse anti-OVA (Sigma Aldrich), mouse anti-LeX (Calbiochem), rat anti-mMGL (ER-MP23; kind gift from Dr. P. Leenen, Erasmus MC, Rotterdam, The Netherlands), rat anti-LAMP1 (BD-Pharmingen), rabbit anti-Rab11 (Life Technologies), goat anti-EEA1 (Santa Cruz Biotechnology) and rabbit anti-EEA-1 (Dianova). Secondary antibodies used: peroxidase-labeled F(ab’)2 fragment goat anti-human IgG, F(ab’)2 fragment goat anti-mouse IgG, (Jackson), peroxidase-labeled goat anti-mouse IgM (Nordic Immunology), goat anti-rat Alexa 448, goat anti-rat Alexa 647, donkey anti-goat Alexa 488, donkey anti-goat Alexa 647, donkey anti-rabbit Alexa 555 and donkey anti-rabbit Alexa 488 (Molecular Probes). MGL-1-Fc was generated as described earlier (Singh et al., 2009b (link)). MR-Fc was kindly provided by L. Martinez-Pomares (University of Nottingham, Nottingham, UK).
8
Polysaccharide Extraction and Characterization from Annona squamosa
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A. squamosa was purchased from Guangzhou Tianhe fruit wholesale market, Guangzhou, China. The pulp material was identified by Professor R.M. Yu, College of Pharmacy, Jinan University, China. Standard monosaccharides and T-series dextrans were obtained from Sigma Chemical Co. (St. Louis, MO, USA). DEAE-52 cellulose and Sephadex G-100 were obtained from Whatman Ltd (Kent County, England). Sephacryl S-300 HR was obtained from Amersham Biosciences (Pharmacia, Uppsala, Sweden). The rmGM-CSF (214-14) and rmIL-4 (315-03) were obtained from PeproTechInc (Rocky Hill, NJ, USA). Anti-CD11c-FITC, anti-MHC II-PE, anti-CD86-FITC and anti-CD11c-APC antibodies were purchased from eBioscience (San Diego, CA, USA). Neutral red was purchased from Amresco (Albany, NY, USA) and the NO assay kit was supplied by the Beyotime Institute of Biotechnology (Haimen, China). Lipopolysaccharide (LPS) and FITC-dextran were purchased from Sigma–Aldrich (St. Louis, MO, USA). Anti-β-actin and anti-GAPDH antibodies were obtained from Biosharp (Beijing, China). Anti-Histone-H3 antibody was obtained from Proteintech (Wuhan, China). MAPK family antibody sampler kit and NF-κB pathway sampler kit were purchased from Cell Signaling Technology (Beverly, MA, USA). Other chemicals and reagents were of analytical grade.
9
Multiparametric Flow Cytometry of Immune Cells
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The following fluorochrome-labelled monoclonal antibodies and staining reagents were used according to manufacturer’s protocols: lamina propria cells were stained with anti-CD11c-APC, anti-CD64-APC (eBioscience), anti-CD11b-PerCP Cy5.5 (Biolegend), anti-CD103-PE/Horizon 421(BD Pharmingen), anti-MHC II-PE-CY7, anti-CD24- PE-CY7 (eBioscience), anti-CD45.2-FITC (eBioscience), anti-CD45.1-Pacific blue (Biolegend) and 4′,6-diamidino-2-phenylindole. TH17 and TH1 cells were stained with anti-IL-17A-PE, anti-CD3-biotin, SA-PerCP, anti-CD4-APC, anti-CD8-FITC, anti-CD45-Pacific Blue anti-IFNγ-PE-CY7 and anti-CD121A-PE. The cells were analysed with LSR Fortessa flow cytometer (BD) or sorted with a FACSAria machine (BD). Flow cytometry analysis was done with the FlowJo software.
10
Multicolor Flow Cytometry Analysis
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Cells (1 × 106) were washed in PBS containing 0.5% BSA. To prevent nonspecific binding cells were incubated with 5% normal rat serum for 10 min at RT. Cells were then stained (45 min, on ice) with the appropriate fluorochrome-conjugated Ab (anti-CD11c-APC, anti-CD11b FITC, anti-CD86 PE, anti-PDCA-1 FITC, anti-Gr-1 PB, anti-MHC-II PE, anti-CD4 PB, anti-CD8 FITC, anti-CD25 PE, and anti-CD3 FITC Ab (eBioscience)). Cells were washed and analyzed using a LSRII cytometer (BD Biosciences). Data analysis was performed with the FlowJo software (version 5.7.2).
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