Primary microglial cells were isolated from the brain via magnetic cell sorting after conjugation with anti-CD11b antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany), as previously described [29 (link)]. Isolated CD11b-positive cells (>90% pure as evaluated by flow cytometry) were stained with anti-CD86-FITC (eBioscience, CA, USA), anti-CD68-APC (BioLegend, CA, USA), anti-TSPO-PE (PBR, Abcam, Cambridge, UK), and anti-CD11b-APC-Cy7 (BD Biosciences, CA, USA) antibodies after treatment with the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences). To measure intracellular cytokines, microglia were plated in poly-d -lysine (PDL)-coated 96-well plates (BD Biosciences) and cultured in DMEM/F-12 (Gibco, CA, USA) medium supplemented with 10% fetal calf serum (Gibco) and 100 U/mL penicillium/streptomycin (Sigma-Aldrich, MO, USA) containing a leukocyte activation-cocktail with BD GolgiPlugTM (BD Biosciences) for 12 h. Microglia cells were treated with the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) and then stained with anti-IL-1β-FITC (eBiosciences), anti-MIP-1α-PE (eBiosciences), anti-TNF-α-APC (BD Pharmingen, CA, USA), and anti-CD11b-APC-Cy7 (BD Biosciences) antibodies. Stained cells were analyzed using a flow cytometer (FACSCanto II; BD Biosciences).
Anti cd11b apc cy7
Manufactured by BD
Sourced in United States, United Kingdom
Anti-CD11b-APC-Cy7 is a fluorescently labeled monoclonal antibody that binds to the CD11b cell surface antigen. CD11b is expressed on the surface of myeloid cells, including monocytes, macrophages, and granulocytes. The APC-Cy7 fluorescent label allows for detection and analysis of CD11b-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (5 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Perc cy5 anti f4 80, by BD (3 mentions)
Anti ly6g pe cy7, by BD (3 mentions)
Bv421 anti siglec f, by BD (3 mentions)
Anti b220 apc, by BD (2 mentions)
Anti cd45 fitc, by BD (2 mentions)
Anti cd5 pe, by BD (2 mentions)
Anti flk1 pe cy7, by BD (2 mentions)
Anti igm per cp cy5, by BD (2 mentions)
Zombie aqua fixable viability kit, by BioLegend (2 mentions)
Facsaria, by BD (2 mentions)
Anti cd11c fitc, by BD (2 mentions)
Anti nk1.1 pe, by BD (2 mentions)
Anti cd45 pe cy7, by BD (2 mentions)
Anti ly6c apc, by BD (2 mentions)
Facscanto, by BD (2 mentions)
Anti cd3 fitc, by BD (2 mentions)
Anti cd25 apc, by BD (2 mentions)
31 protocols using anti cd11b apc cy7
1
Isolation and Characterization of Primary Microglia
2
Monocyte and DC Recruitment in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze monocyte and DC recruitment in response to CpG, 20 µl of a 100 µg/ml ODN1864 solution was injected into the footpad of C57BL/6 J mice. At the indicated time intervals post injection, blood samples were collected through the tail vein followed by ACK (Thermo Fisher Scientific) mediated red blood cell lysis. Alternatively, mice were euthanized and the DLNs were dissected. Single cell suspensions of DLN were prepared through incubation with collagenase type IV (Sigma-Aldrich) and passed through a 70 µm cell strainer (BD Falcon).
After treatment with 2.4G2 Ab for 5 min to block the FcR, PBMCs or lymph node cell suspensions were stained with the following anti-mouse Abs: anti-CD45-V450, anti- MHCII-FITC, anti-CD86-PE, anti-CD40- PE, anti-CD64-APC, anti-F4/80-APC, anti-Ly6C-PE-Cy7, anti-CD11b-APC-Cy7 (all BD Biosciences), Ly6G-PerCP-Cy5.5, CCR2-APC (eBioscience), CD64-BV421 (BioLegend) and CD11c-PE-TxR (R & D systems). Death cells were identified by staining with aqua life/dead stain (Invitrogen). Fluorescent events were acquired using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software. After exclusion of dead cells, granulocytes were identified as CD45+ CD11b+ Ly6C+ Ly6G+ cells and Monocytes as CD45+ Ly6Chi CD11bhi Ly6G- cells. Following exclusion of granulocytes and Monocytes in the LN, DCs were subdivided into MHCIIhi DCs and MHCIIint DCs.
After treatment with 2.4G2 Ab for 5 min to block the FcR, PBMCs or lymph node cell suspensions were stained with the following anti-mouse Abs: anti-CD45-V450, anti- MHCII-FITC, anti-CD86-PE, anti-CD40- PE, anti-CD64-APC, anti-F4/80-APC, anti-Ly6C-PE-Cy7, anti-CD11b-APC-Cy7 (all BD Biosciences), Ly6G-PerCP-Cy5.5, CCR2-APC (eBioscience), CD64-BV421 (BioLegend) and CD11c-PE-TxR (R & D systems). Death cells were identified by staining with aqua life/dead stain (Invitrogen). Fluorescent events were acquired using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software. After exclusion of dead cells, granulocytes were identified as CD45+ CD11b+ Ly6C+ Ly6G+ cells and Monocytes as CD45+ Ly6Chi CD11bhi Ly6G- cells. Following exclusion of granulocytes and Monocytes in the LN, DCs were subdivided into MHCIIhi DCs and MHCIIint DCs.
3
Isolation of Microglia from Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were perfused with PBS containing 5 IU/ml heparin. Isolated brains were stored on ice in HBSS with 45% glucose and HEPES and subsequently minced with a scalpel and dissociated in high-glucose DMEM containing collagenase A, FCS, and DNAse I (30 minutes, 37°C). Cells were centrifuged (10 min, 400g, 4˚C) and resuspended in 5 ml of 25% Percoll overlaid with 3 ml of ice-cold PBS. Microglial cells were obtained as a pellet after centrifugation (30 min, 800g, 4°C), and resuspended in FACS buffer (0.5% BSA, 2mM EDTA in PBS). Microglia were stained (30 min, on ice, in the dark) using anti-CD45-PE (1:800; clone 30-F11, eBioscience), anti-CD11b-APC-Cy7 (1:400; clone M1/70, BD Biosciences), and Fc receptor blocking antibody CD16/CD32 (1:400; clone 2.4G2, BD Biosciences). Microglia were sorted as CD45int CD11b+ cells with an Aria II (BD Biosciences) to >90% purity.
4
Spleen Cell Fractionation and Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleen cells were stained with anti-CD45-FITC (BD Bioscience, Franklin Lakes, USA), anti-sca1-BV421 (BD Bioscience, Franklin Lakes, USA), anti-flk1-PE-Cy7 (BD Bioscience, Franklin Lakes, USA), anti-B220-APC (BD Bioscience, Franklin Lakes, USA), anti-IgM-Per-CP/Cy5.5 (BD Bioscience, Franklin Lakes, USA), anti-CD11b-APC/Cy7 (BD Bioscience, Franklin Lakes, USA) and anti-CD5-PE (BD Bioscience, Franklin Lakes, USA). Dead cells were identified by using a Zombie Aqua™ Fixable Viability Kit (Biolegend, San Diego USA) and doublets were excluded.
The following fractions were sorted by using flow cytometry (FACSAria, BD): CD45+, sca1+/flk1+ (sca1+/flk1+ cells); CD45+, B220+/IgM+, CD11b−/CD5− (B2 cells); CD45+/sca1−/flk1−, B220+/IgM+, CD11b−/CD5− (sca1−/flk1− B2 cells).
The following fractions were sorted by using flow cytometry (FACSAria, BD): CD45+, sca1+/flk1+ (sca1+/flk1+ cells); CD45+, B220+/IgM+, CD11b−/CD5− (B2 cells); CD45+/sca1−/flk1−, B220+/IgM+, CD11b−/CD5− (sca1−/flk1− B2 cells).
5
Phenotypic Characterization of Peritoneal Cells during Toxoplasma Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
On the third day of infection, peritoneal cells from WT mice infected with 1 × 107 Nc-1 tachyzoites were collected and stained for phenotypic characterization and Dectin-1 expression. Briefly, mice were euthanized, and their peritoneal cavities were washed with ice-cold PBS. The suspension was then centrifuged at 400 × g, at 4°C, for 10 min. The cell pellet was resuspended in PBS with 5% of normal rabbit serum, at room temperature, for 15 min, prior to incubation with the appropriate antibodies: anti-CD11b-APC-Cy7, anti-CD11c-FITC, anti-MHCII-PE, anti-CD11c-V450, anti-CD19-APC-Cy7, anti-CD3-Pacific Blue, anti-CD49b-APC (BD Biosciences), and anti-Dectin-1-PE (R&D Systems, Minneapolis, MN, USA). Cells were incubated with primary antibodies conjugated to the different fluorochromes for another 30 min, at room temperature. After washing, cells were suspended in PBS with 3% formaldehyde, read by flow cytometry (FACSCantoII, BD Biosciences), and analyzed using dedicated software (FlowJo X, Tree Star Inc., Ashland, OR, USA).
6
Isolation and Characterization of Immune Cells from Mouse Spinal Cord
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
7
Multiparametric flow cytometry for immune cells
8
Multiparametric Flow Cytometry of Mouse MNCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse MNCs were stained with the following fluorescence-conjugated surface mAbs: anti-CD3-FITC, anti-CD4-APC, anti-CD8-PE-cy7, anti-NK1.1-PE, anti-CD11b-APC-cy7, anti-CD103-BV421, anti-CD11c-BV510, and anti-CD25-APC antibodies (BD PharMingen, USA). The cells were stained according to the standard procedure of BD PharMingen. Fixation and permeabilization were performed using the Transcription Factor Buffer Set (BD PharMingen, USA), and the cells were then stained with the anti-FoxP3-BV421 intracellular antibodies for 40 min at 4°C. To detect Th1 and Th17 cytokines, MNCs suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum were stimulated with a cell stimulation cocktail (eBioscience, USA) containing phorbol-12-myristate-13-acetate (PMA, 50 ng/mL), ionomycin (1 μg/mL), and monensin (2 μg/mL) at 37°C with 5% CO2 for 6 h, and followed by intracellular staining with fluorescently labeled anti-IFN-γ and anti-IL-17 antibodies (BioLegend, USA) after washing, fixing, and permeabilizing according to the manufacturer's instructions. Isotype IgGs were used as a control. All samples were detected using a BD LSR Fortessa X-20 Flow Cytometry System (BD, USA) and analyzed using the FlowJo software.
9
Quantifying Myeloid Subsets in 5TGM1 Tumors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total BM was collected from naïve or 5TGM1 tumour‐bearing KaLwRij mice at experimental end‐points, and 1 × 106 cells were stained with mouse monoclonal anti‐CD11b APC‐Cy7 (BD Biosciences), anti‐Ly‐6C BV421 and anti‐Ly‐6G PE‐Cy7 antibodies (Biolegend) and subjected to analysis by flow cytometry using the LSRFortessa X‐20 flow cytometer (BD Biosciences).
10
Monocyte Isolation and Cytokine Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For ex vivo stimulation and cytokine production, monocytes were isolated from PBMC (EasySep Human monocytes enrichment kit w/o CD16, StemCell, Vancouver, Canada), according to manufacturer instructions. Purified monocyte fraction was stained with anti-CD14-FITC (BD Bioscience, San Jose, CA, USA) and anti-CD11b-APC-Cy7 (Beckman Coulter, Brea, CA, USA) and purity was evaluated via FC. Purified monocyte fraction was plated in 96-wells flat bottom plate (105 monocytes/well) and incubated for 1 h in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 1% L-glutamine and 1% sodium-pyruvate at 37 °C. After that, culture media was removed, and cells were washed with warmed PBS as extra of purification. New warmed culture media was combined with 10 ng/mL of LPS and monocytes were incubated overnight. Supernatant was collected and stored at −20 °C.
For RNA extraction, monocytes were isolated from PBMC (CD14 microbeads, MiltenyiBiotec, Bergisch Gladbach, Germany), according to manufacturer instructions. Purified monocytes were stained with anti-CD14-FITC (BD Bioscience, San Jose, CA, USA) and anti-CD11b-APC-Cy7 (BC), and purity was evaluated via FC. Purified monocytes (purity > 90%) were used to extract RNA.
For RNA extraction, monocytes were isolated from PBMC (CD14 microbeads, MiltenyiBiotec, Bergisch Gladbach, Germany), according to manufacturer instructions. Purified monocytes were stained with anti-CD14-FITC (BD Bioscience, San Jose, CA, USA) and anti-CD11b-APC-Cy7 (BC), and purity was evaluated via FC. Purified monocytes (purity > 90%) were used to extract RNA.
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!