For multicolor flow cytofluorimetric analysis, PBMCs, LMNCs and PMNCs were stained with the following conjugated mAb as previously described: CD56-PE-Cy5 and NKp46-PE (Beckman Coulter, clone BAB281), CD16-PE-Cy7, CD19-APCH7, IFN-γ-PE, DNAM-1 PE, CD49e-PE, CD11c-PE, CCR5-PE, CD69-PE, CCR4-PE and CXCR3-PE (BD-Pharmigen), CD45-PB, CD62L-PE-Cy7, CCL5-AF647, CD161-PerCpCy5.5, CCR3-PE, CXCR2-PE and CXCR4-PerCpCy5.5 (Biolegend), CCR7-FITC and CXCR6-PE (R&D System), CX3CR1 PE (MBL). Aqua LIVE/DEAD (Life Technologies) was used to eliminate dead cells from the analysis. Intracellular staining was performed using Cytofix/Cytoperm (Beckton Dickinson), according to the manufacturers instructions. The gating strategy used to select NK cells from both PBMC and LMNC is depicted in Supplemental Figure 1 . For measurement of IFN-γ production, whole PBMC and LMNC were stimulated with 18 hours with 20ng/ml rhIL-12 (R&D Systems) and 200U/ml rhIL-2 (Peprotech). For the final 4 hours, GolgiStop was added (Beckton Dickenson). Flow cytometry data was acquired using an LSR Fortessa (Beckton Dickinson) and data was analyzed using FlowJo Software (Tree Star).
Cd11c pe
Manufactured by BD
Sourced in United States
CD11c-PE is a fluorescently labeled monoclonal antibody that binds to the CD11c antigen, which is expressed on the surface of certain immune cells, such as dendritic cells and monocytes. This product is commonly used in flow cytometry applications to identify and quantify these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 pe, by BD (7 mentions)
Cd11b apc, by BD (7 mentions)
Cd80 fitc, by BD (6 mentions)
Cd86 fitc, by BD (5 mentions)
Cytofix cytoperm, by BD (4 mentions)
Collagenase, by Merck Group (4 mentions)
F4 80 apc cy7, by BioLegend (4 mentions)
Sytox blue, by Thermo Fisher Scientific (4 mentions)
F4 80 apc, by Thermo Fisher Scientific (4 mentions)
Lsrfortessa, by BD (3 mentions)
Hla dr percp, by BD (3 mentions)
Cd11b fitc, by BD (3 mentions)
Cd45 percp cy5, by BD (3 mentions)
Cd4 fitc, by BD (3 mentions)
Cd11c fitc, by BD (3 mentions)
Cd45 pe cy7, by Thermo Fisher Scientific (3 mentions)
Cd206 alex647, by Bio-Rad (3 mentions)
Sytox blue, by Tree Star (3 mentions)
Facsaria cell sorter, by BD (3 mentions)
Foxp3 pe, by BD (3 mentions)
57 protocols using cd11c pe
1
Multicolor Flow Cytometry Analysis of PBMC, LMNC and PMNC
2
Differentiation of THP-1 Monocytes into Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The acute monocytic human leukemia cell line THP-1 cells (ATCC® TIB-202™) were cultured in RPMI 1640 medium supplemented with 10 mM HEPES, 2 mM L-glutamine, 25 mM D-glucose, 1 mM sodium pyruvate (Gibco, Thermo Fisher Scientific) and 10% fetal bovine serum (Sigma-Aldrich) at 37°C with 5% CO2 and were passaged every 3–4 days. Cells were differentiated into macrophages by incubation with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 3 days, followed by 5-day rest in a medium without PMA. The differentiation protocol was selected based on preliminary results in which THP-1 monocytes were differentiated into macrophages with different PMA concentrations (30, 100, and 162 nM) and for different periods of time (24 h and 1 day rest, 72 h and 1 day rest and 24 or 72 h and 5 day rest), and then the expression of CD11c (CD11c PE, BD Pharmigen), CD14 (CD14 PerCP-Cy5.5, BD Pharmigen), CD16 (CD16 APC, BD Pharmigen), CD68 (CD68 FITC, BioLegend), CD163 (CD163 PE, Invitrogen) and viability (7-AAD, BD) were measured by flow cytometry and confocal microscopy.
3
Monocyte-Derived Dendritic Cell Activation by HSPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monocytes were isolated by human monocyte enrichment cocktail (Stemcell Technologies, France). Isolated monocytes were cultured with recombinant human granulocyte macrophage colony‐stimulating factor and interleukin‐4 (IL‐4) (Immunotools, Germany), 50 ng/mL of each in RPMI complete media. At day 3, half of the media was replaced with fresh RPMI complete media with granulocyte macrophage colony‐stimulating factor and IL‐4. At day 6, 2×106/mL cells were cultured with or without HSP60 (MyBiosource) or HSP90 (HSPß90, Sigma Aldrich, MO) at the concentrations described. Cells were collected after 24 hours and analyzed with >90% cell viability. DC stained with CD11C‐PE, CD86‐Percp/Cy5.5, CD83‐APC, CD40‐fluorescein isothiocyanate (BD Bioscience), and HLA‐II‐fluorescein isothiocyanate (Biolegend) antibodies.
The endotoxin concentration in HSP60 was checked by LAL Chromogenic Endotoxin Quantitation Kit (Thermofisher Scientific, IL). The level of endotoxin in the HSP60 was ≤1 EU/mg, according to manufacturer information, which is equal to ≤0.1 ng/mg. Our working concentration 5 μg/mL of HSP60 contains ≤0.0005 ng endotoxin. A similar concentration of endotoxin was determined in HSP90.
The endotoxin concentration in HSP60 was checked by LAL Chromogenic Endotoxin Quantitation Kit (Thermofisher Scientific, IL). The level of endotoxin in the HSP60 was ≤1 EU/mg, according to manufacturer information, which is equal to ≤0.1 ng/mg. Our working concentration 5 μg/mL of HSP60 contains ≤0.0005 ng endotoxin. A similar concentration of endotoxin was determined in HSP90.
4
Analysis of moDC surface markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine the expression of surface markers on moDC (1 × 106 cells/ml) the cells were incubated with primary antibodies on ice for 30 min. Antibodies used for flow cytometry included HLA-DRperCP, CD11cPE, CD14PEcy7 (BD Biosciences, USA). Data were acquired by flow cytometry on a LSR II instrument (BD biosciences) and analyzed using FlowJo (Tree star, USA).
5
Investigating YS Effects on DC Phenotype
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analysis the effects of YS on the DCs mutation, day 7 bone marrow derived DCs (105/mL) were incubated with YS-OVA containing 2 ug OVA or 2 ug soluble OVA. Then cells were subsequently stained with CD11c-PE (BD Pharmingen), MCH-II-FITC (BD Pharmingen), CD80-FITC (BD Pharmingen) and CD86-FITC (BD Pharmingen). These surface markers levels was measured by flow cytometry (BD Immunocytometry Systems) and analyzed by FlowJo software Version 6 (TreeStar). DCs culture supernatants were collected 24 h after culture with samples. The IL-12 cytokine production in the media was analyzed using enzyme-linked immunosorbant assay (ELISA) kits (Becton Dickinson, NJ, USA) according to manufacturer’s directions.
6
Analyzing Qβ-Specific B Cells in Germinal Center
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens of immunized mice were harvested at indicated time points and single cell suspensions were obtained by passing through 70 μm cell strainer (Greiner Bio-ONE, cat. 542070). Single cells were suspended in FACS buffer (2% FBS in PBS) and incubated with antibody at 4°C. To distinguish the Qβ-specific B cells in germinal center, cells were firstly stained with Qβ-AlexaFluoro 488 and PNAbio (Vector Laboratories, cat. B-1075), and then Fc-receptors were blocked with anti-mouse CD16/CD32 (BD Bioscience, cat. 553142). Finally, streptavidin-APC Cy7 (BD Bioscience, cat. 554063) and anti-mouse CD38-PerCP Cy5.5 (Biolegend, cat. 102722) were applied to determine germinal center cells, anti-mouse B220-PE Cy7(BD Bioscience, 552772) for B cells, anti-mouse IgM-PE (Jackson ImmunoResearch, cat. 115-116-075), IgD-PE (eBioscience, cat. 12-5993-83), CD4-PE (BD Bioscience, cat. 553653), CD8-PE (BD Bioscience, cat. 553032), CD11b-PE (BD Biosience, cat. 553311), CD11c-PE (BD Biosience, cat. 553802), GR1-PE (BD Biosience, cat. 553128) to exclude other cell types (CD4, CD8: T cells; CD11b: monocytes and macrophages; CD11c: dendritic cells; GR1: neutrophils) and immature B (IgM+IgD+) cells.
7
Quantification of BTLA on mDCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of BTLA on mDCs was determined by ow cytometry. Monoclonal antibodies Lin1-FITC, HLA-DR-PerCP, CD11c-PE and BTLA-APC were purchased from BD Biosciences (San Diego, CA, USA). 100µL fresh collected whole blood of patients or controls was incubated with indicated antibodies for 30minutes at 4 °C and then lysed with FACS™ lysing solution (BD Biosciences, San Jose, CA, USA).Subsequently, those stained samples were washed with phosphate buffered saline (PBS), xed and eventually detected by BD FACS calibur with BD CellQuest software supporting. Data were analyzed using FlowJo software 7.6 (Tree Star Inc., San Carlos, CA, USA).
8
Comprehensive Immune Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monoclonal antibodies CD3e-ApcCy7, CD45-PerCPCy5.5, Gr1-APC, CD8-PECy7, CD25-FITC, CD11b-FITC, CD11C-PE, B220-PB, and NK1.1-PE were purchased from BD Biosciences. Foxp3-APC and F4/80-PE were purchased from eBioscience and CD4-PO and calcein violet from Invitrogen.
9
Immune Cell Profiling of Polyp Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the immune cell profile of whole polyp cells, fresh and cultured (for 72 hours) cells were harvested (both adherent and nonadherent cells) without enzymatic dissociation and labeled with a set of various specific fluorescent antibodies, such as CD4-Pacifc Blue, CD8-PECy7, CD11c-PE, CD14-PerCPCy5.5, CD19-Viollet, CD25-APC, CD40-FITC, CD69-FITC, and NK1.1-PE (all from BD Biosciences). These sets of antibodies were used for both fresh and in vitro coculture analysis. To investigate the CD4+CD25+Foxp3+ T lymphocyte profile, intracellular staining for FoxP3 expression was performed after 72 hours in culture with or without the presence of MSCs. The cells were fixed and permeabilized using the Fix/Perm Buffer Set Kit (BD Biosciences). Staining was performed using FoxP3 (PE), CD4 (APC-Cy7), and CD25 (APC) antibodies at 1 : 100 dilution (BD Biosciences). Analysis was determined by flow cytometry (FACSCanto II cytometer, BD Biosciences) within FSC and SSC gate in CD4+ T cells. All acquisitions were performed using a FACSCanto II flow cytometer (BD Biosciences), and analysis was again done using the FlowJo 7.2.4 software (Tree Star). During analysis, gates for mononuclear cells were performed in FSC and SSC parameters after doublet exclusion. Results are presented as cell frequency.
10
Single-Vesicle Detection of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Amnis® CellStream instrument (Luminex, US) was used to detect EVs at single‐vesicle level. EVs without fluorescent tags were first stained with 0.4 μl of sLeX antibodies (diluted by 10‐fold) overnight. The stained and mNG‐labelled EVs were analyzed using the side scatter laser (SSC; 40% of maximum power), 488 nm laser (maximum power) and 642 nm laser (maximum power). All data were acquired at a flow rate of 3.44 μl/min. SSC and AF647 signals were collected in channel 1 (773 ± 56 nm filter) and channel B6 (702 ± 87 nm filter), respectively. AF488 and mNG signals were collected in channel C3 (528 ± 46 nm filter). DPBS (Gibco, 14190‐136) was used as sheath fluid without further filtration. Data were analyzed with optimized masking settings using the FlowJo software.
The following antibodies and corresponding isotypes were used: sLeX‐AF488 (BD, 563528), sLeX‐AF647 (BD, 563526), LeX‐APC (BD, 561716), CD11c‐PE (BD, 557401), F4/80‐BV510 (BD, 743280), DC‐SIGN‐PE (BioLegend, 833004), and E‐selectin‐APC (BD, 551144).
The following antibodies and corresponding isotypes were used: sLeX‐AF488 (BD, 563528), sLeX‐AF647 (BD, 563526), LeX‐APC (BD, 561716), CD11c‐PE (BD, 557401), F4/80‐BV510 (BD, 743280), DC‐SIGN‐PE (BioLegend, 833004), and E‐selectin‐APC (BD, 551144).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!