Dopamine hydrochloride (DA), tris(hydroxymethyl)aminomethane (TRIS), Streptavidin (SA), WST-1 Cell Proliferation Reagent, ammonium hydroxide solution (NH4OH, 25 %), gentamicin, Poly-L-lysine solution (0.1 % w/v in H2O) and 2-mercaptoethanol were purchased from Sigma-Aldrich. mPEG-SH (2 kDa) and biotin-PEG-SH (2 kDa,) were purchased from NANOCS. RPMI 1640 was purchased from Lonza. L-glutamine was purchased from Gibco. 10 % heat-inactivated fetal calf serum was purchased from BioScience. GM-CSF was purchased from Peprotech. Anti-mouse CD11c-FITC was purchased from eBioscience. Poly-L-lysine was purchased from Invitrogen. Fluorescent nanodiamonds were supplied by Adamas Nanotechnologies and Columbus NanoWorks. Deionized (DI) water with a resistivity of 18.2 MΩ·cm was from Milli-Q Water Purification System.
Anti mouse cd11c fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-mouse CD11c-FITC is a fluorochrome-conjugated monoclonal antibody that binds to the CD11c antigen expressed on the surface of mouse dendritic cells. It can be used for the identification and analysis of dendritic cells in flow cytometry applications.
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Lab products found in correlation
Anti mouse cd11b apc, by Thermo Fisher Scientific (2 mentions)
Anti mouse mhc 2 fitc, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Anti mouse cd86 pe, by Thermo Fisher Scientific (2 mentions)
Ammonium hydroxide solution, by Merck Group (1 mentions)
Poly l lysine (pll), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Cell proliferation reagent wst 1, by Merck Group (1 mentions)
Mpeg sh, by Nanocs (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Poly l lysine solution, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Streptavidin, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Dopamine hydrochloride, by Merck Group (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Biotin peg sh, by Nanocs (1 mentions)
Anti mouse cd25 pe, by Thermo Fisher Scientific (1 mentions)
Anti mouse foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd4 apc, by Thermo Fisher Scientific (1 mentions)
10 protocols using anti mouse cd11c fitc
1
Nanoparticle-based Cell Proliferation Assay
2
Soybean Bioactive Compound Extraction
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The soybean ‘Dongnong 42’ was provided by the Northeast Agricultural University. MTG (food additive, enzyme activity: 102.3 U/mL) was provided by Taixing Yiming Biological Products Co., Ltd. (Taixing, China). The anti-mouse CD11c−FITC, anti-mouse MHCII−FITC, anti-mouse CD4−APC, anti-mouse CD25−PE, anti-mouse IL-4−BV421, anti-mouse IFN-γ−Percp, and anti-mouse Foxp3−APC were also purchased from eBioscience, Inc. (San Diego, CA, USA). All reagents were of analytical grade.
3
Murine Myeloid-Derived Suppressor Cell Analysis
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Bone marrow (BM) cells were flushed from mouse femurs and tibiae and prepared as single-cell suspension after lysing the red blood cells. Peripheral blood mononuclear cells (PBMC) in mouse blood samples were isolated with Ficoll-Hypaque (Axis-Shield). All cells labeled with antibodies were immediately analyzed on a FACS Calibur (BD Biosciences, Mountain View, CA, USA).
For the analysis of MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, and anti-mouse Gr1-PE.
For the analysis of PMN-MDSCs and M-MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, anti-mouse Ly6G-FITC, and anti-mouse Ly6C-PE.
For the analysis of immature or undifferentiated markers CD11c, F4/80, CD80, and MHCII on MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, anti-mouse Gr1-PE or anti-mouse Gr1-FITC, and anti-mouse CD11c-FITC, anti-mouse F4/80-FITC, anti-mouse CD80-PE, or anti-mouse MHCII-PE.
For the analysis of MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, and anti-mouse Gr1-PE.
For the analysis of PMN-MDSCs and M-MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, anti-mouse Ly6G-FITC, and anti-mouse Ly6C-PE.
For the analysis of immature or undifferentiated markers CD11c, F4/80, CD80, and MHCII on MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, anti-mouse Gr1-PE or anti-mouse Gr1-FITC, and anti-mouse CD11c-FITC, anti-mouse F4/80-FITC, anti-mouse CD80-PE, or anti-mouse MHCII-PE.
4
Evaluating Antigen Cross-presentation in BM-DCs
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For the evaluation of antigen cross-presentation, WT and P2X7KO BM-DCs were stimulated for 24 h with H-AC-OVA and H-ACs-OVA-P2X7, in a 2:1 ratio of ACs/BM-DCs. As a positive control, BM-DCs were incubated with 5 μM of OVA257-264 peptide (SIINFEKL, Sigma-Aldrich, St. Louis, MO, USA) for 180 min at 37° C in a 5% CO2 atmosphere. After the pulse period, BMDCs labeling was performed with the anti-mouse- CD11c FITC and anti-mouse OVA antibodies (SIINFEKL/H-2Kb, clone eBio25D1.16 APC) from, both from eBioscience, San Diego, CA, USA. Total viable cells were evaluated by PI + exclusion. SIINFEKL/MHC-I complex was analyzed in the BM-DCs surface using the Accuri C6 Flow Cytometer and the information was processed using CFlow Plus software (eBioscience, San Diego, CA, USA)
5
Immune Cell Subset Analysis in Murine Splenocytes
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Splenocytes were obtained from the spleen obtained from autopsy at age 16 weeks, and were stained with fluorescence labeled cell surface antibodies to analyze immune cell subsets. Cells which were surface stained, were analyzes with FACSAria III (BD Bioscience, San Jose, CA, USA). Cell surface staining was performed with antibodies as follow: Anti-mouse CD4 PE-Cyanine5 (#15-0042), Anti-mouse CD5 APC-eFluor® 780 (#47-0051), Anti-mouse CD8a FITC (#11-0081), Anti-mouse Ly-6G (Gr-1) FITC (#11-5931), Anti-mouse Ly-6C APC (#17-5932), Anti-mouse CD11b APC (#17-0112), Anti-mouse CD11c FITC (#11-0114), Anti-mouse CD11c PE-Cyanine5 (#15-0114), Anti-mouse CD11c Alexa Fluor® 700 (#56-0114), Anti-mouse CD19 PE-Cyanine5 (#15-0193), Anti-mouse CD19 PE-eFluor® 610 (#61-0193), Anti-mouse CD19 Alexa Fluor® 700 (#56-0193), Anti-mouse NK1.1 PE-eFluor® 610 (#61-5941) and Anti-mouse MHC Class II (I-A/I-E) PE-Cyanine7 (#25-5321), all from eBioscience (San Diego, CA, USA).
6
Fura-2 AM and ORAI1-CT Calcium Signaling
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Fura-2 AM calcium indicator was purchased form Life Technologies (Carlsbad, CA, USA). Phorbol 12-myristate 12-acetate (PMA), ionomycin, thapsigargin (TG) and isopropyl-ginethiogalactopyranoside (IPTG) were purchased from Sigma Aldrich (St Louis, MO, USA). Tri(2-carboxyethyl)phosphine (TCEP) was obtained from Pierce (Life Technologies). Amylose resin used for MBP pulldown was purchased from New England Biolabs (Ipswich, MA, USA). Ni-NTA resin used for purification of GB1-ORAI1-CT was purchased from Qiagen (Valencia, CA, USA). The mouse monoclonal anti-Flag M2-HRP (A859, Sigma-Aldrich, St. Louis, MO, USA) antibody, the rabbit anti-mCherry polyclonal antibody (NBP2-25157, Novus Biologicals, Littleton, CO, USA), the rabbit anti-Caspase-1 antibody (D7F10, Cell signaling, Danvers, MA, USA) and the rabbit anti-IL-1β antibody (sc-7884, Santa Cruz Biotechnology, Dallas, TX, USA) were used at a 1:1000 dilution. For flow cytometry (FACS) analysis, anti-mouse MHC Class II (I-A/I-E) APC (), anti-mouse IFN gamma PE (), anti-mouse CD86 (B7-2) PE(12–7311), anti-mouse CD197 (CCR7) PE (12–1971), anti-mouse MHC Class I PE (12–9558), anti-mouse CD11c FITC (11–0114), anti-mouse CD4 PerCP-Cyanine5.5 (45–0042), and anti-mouse CD8a APC (17–0081) were purchased from eBioscience. All other reagents were form Sigma-Aldrich unless otherwise indicated.
7
Immunophenotyping of Tumor-Challenged Mice
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Tumor-challenged mice were treated as described earlier, and on day 10 after the first treatment, mice from each group (n=3) were euthanized and the cells from the spleens, tumor-draining lymph nodes (TDLNs), and tumors were surgically removed and used for MDSC, Treg, DC, and CD4+ and CD8+T-cell quantification by flow cytometry. A single cell suspension of spleens and TDLNs was prepared by filtration through a 300-gauge mesh. Tumor suspensions were prepared as described previously, with modifications.28 (link) The cell suspensions were then stained at 4 degree for 30 min using the following antibodies: FITC anti-mouse CD11b, PE anti-mouse Gr-1, FITC anti-mouse CD4, PE anti-mouse FOXP3, FITC anti-mouse CD11c, PE anti-mouse CD86, PE-cy5 anti-mouse CD3, FITC anti-mouse CD4, PE anti-mouse CD8, and the corresponding isotype control antibodies (all monoclonal antibodies were obtained from eBioscience). After washing with PBS, the cells were fixed with 10% formaldehyde. The cell frequency was determined using BD FACSCalibur. The samples were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).
8
Trichinella spiralis Infection Murine Model
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Trichinella spiralis (ISS534) were obtained from specific pathogen-free female inbred Wistar rats (220.0 ± 20.0 g), maintained by serial oral passage. Eight-week-old BALB/c and C57BL/6 male mice (22.0 ± 2.0 g) were housed under proper care and specific pathogen-free conditions by the institutional guidelines. Wistar rats, BALB/c and C57BL/6 mice were purchased from Jilin University Experimental Animal Center (Jilin, China), with certificate No. 2016-0001 in conformity with SCXK (Jilin). The H22 hepatoma cell line was purchased from Bio-Rad Life Sciences Development Co., Ltd. (Beijing, China). FITC anti-mouse CD11c, PE Anti-Mouse MHC Class II (I-A), and PE anti-mouse CD86 were purchased from eBioscience (USA).
9
Flow Cytometry Analysis of DC Activation
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The surface markers expressed in the stimulated DCs were assessed by using a FACS method. The proportion of DCs expressing co-stimulatory surface markers was determined in CD11c-gated DCs. DCs (1 × 106) pulsed with STG, LPS, or PBS as a negative control were stained with the following monoclonal antibodies: FITC anti-mouse CD11c (eBioscience, USA; clone N418e), APC anti-mouse MHC-II (eBioscience; clone M5/114.15.2), and CD80 and PE anti-mouse CD40 (eBioscience; clone 1C10). The stained cells were washed three times with FACS running buffer (Miltenyi Biotec, Germany). Subsequently, the antigen-pulsed DCs were sorted with a MACSQuant Analyzer (Miltenyi Biotec).
10
Murine Dendritic Cell Phenotyping
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Following purification, MLNDCs were cultured in RPMI-1640 with 10% fetal bovine serum, 100 IU/mL penicillin and 100 ug/mL streptomycin and incubated at 37°C, 5% CO2.
Reagents: phosphate buffered saline (PBS; GNM-20012, Hangzhou, China), RPMI1640 and 10% fetal bovine serum (Gibco, Grand Island, NY, USA), PE-anti-mouse CD11c, APC-anti-mouse MHC-I, FITC-anti-mouse MHC-II, FITC-anti-mouse CD80, APC-anti-mouse CD86, PE-mouse IgG1 control, APC-mouse IgG2a control, FITC-mouse IgG2b control (eBioscience, Sandiego, California, USA), 4% paraformaldehyde (Sigma, St. Louis, Missouri, USA), blocking solution (Sigma), Anti-CRHR1 (Anbo Biotechnology, San Francisco, California, USA), Anti-CRHR2 (Anbo Biotechnology), FITC anti-Mouse CD11c (eBioscience), Cy3 conjugated goat anti-rabbit IgG (H+L), 4′,6-diamidino-2-phenylindole (DAPI) (Southern Biotech Associates, Birmingham, Alabama, USA), anti-fading agent (Sigma). Ten percent of SDS-PAGE (Amersham Biosciences, Piscataway, New Jersey, USA), polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences), goat anti-rabbit MHC-II (Abcam, Cambridge, UK), HRP-conjugated secondary antibody(Abcam). Carboxyfluorescein diacetate succinimidyl ester (Invitrogen Ltd, Paisley, UK), anti-mouse CD4 antibody, and anti-mouse CD8 antibody (Invitrogen Ltd).
Reagents: phosphate buffered saline (PBS; GNM-20012, Hangzhou, China), RPMI1640 and 10% fetal bovine serum (Gibco, Grand Island, NY, USA), PE-anti-mouse CD11c, APC-anti-mouse MHC-I, FITC-anti-mouse MHC-II, FITC-anti-mouse CD80, APC-anti-mouse CD86, PE-mouse IgG1 control, APC-mouse IgG2a control, FITC-mouse IgG2b control (eBioscience, Sandiego, California, USA), 4% paraformaldehyde (Sigma, St. Louis, Missouri, USA), blocking solution (Sigma), Anti-CRHR1 (Anbo Biotechnology, San Francisco, California, USA), Anti-CRHR2 (Anbo Biotechnology), FITC anti-Mouse CD11c (eBioscience), Cy3 conjugated goat anti-rabbit IgG (H+L), 4′,6-diamidino-2-phenylindole (DAPI) (Southern Biotech Associates, Birmingham, Alabama, USA), anti-fading agent (Sigma). Ten percent of SDS-PAGE (Amersham Biosciences, Piscataway, New Jersey, USA), polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences), goat anti-rabbit MHC-II (Abcam, Cambridge, UK), HRP-conjugated secondary antibody(Abcam). Carboxyfluorescein diacetate succinimidyl ester (Invitrogen Ltd, Paisley, UK), anti-mouse CD4 antibody, and anti-mouse CD8 antibody (Invitrogen Ltd).
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