Thymocytes from a C57BL/6N mouse were treated with 0.1 μM dexamethasone (Sigma) for 14 hr at 37°C to induce apoptosis and then stained with pHrodo Red, succinimidyl ester (ThermoScientific) for 1 hr. Fully differentiated BMDMs from WT and SIRPA KO mice (2 mice each) were incubated in duplicate with 1mM MnCl2 (SIGMA) and fed apoptotic thymocytes at a ratio of 1:5 for 2 hr at 37°C. The cells were detached with 1mM EDTA, stained with APC-conjugated anti-CD11b (Biolegend) for 1hr at 4°C and analyzed by FACS. The percentage of double-positive cells was determined, and the percent of engulfment was normalized to WT cells for each experiment. Presented is the average of 2 independent experiments.
Apc conjugated anti cd11b
Manufactured by BioLegend
Sourced in United States
APC-conjugated anti-CD11b is a monoclonal antibody that binds to the CD11b cell surface antigen. CD11b is expressed on the surface of various immune cells, including monocytes, macrophages, and neutrophils. This antibody is labeled with the fluorescent dye Allophycocyanin (APC), which can be detected using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Phrodo red succinimidyl ester, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Mncl2, by Merck Group (1 mentions)
Percp conjugated anti cd45, by BioLegend (1 mentions)
Pe cy7 conjugated anti cd11c, by BioLegend (1 mentions)
Trustain fcx, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Anti cd86, by BioLegend (1 mentions)
Anti cd80, by BioLegend (1 mentions)
Anti cd69, by BioLegend (1 mentions)
Fluorescein isothiocyanate conjugated anti cd45, by BioLegend (1 mentions)
Pe conjugated anti cd4, by BioLegend (1 mentions)
Anti cd11b, by BioLegend (1 mentions)
Apc conjugated anti cd19, by BD (1 mentions)
Pe conjugated anti cd23, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti igm, by BD (1 mentions)
Bv605 conjugated streptavidin, by BioLegend (1 mentions)
Fitc conjugated anti cd21, by BD (1 mentions)
16 protocols using apc conjugated anti cd11b
1
Dexamethasone-induced Thymocyte Apoptosis Phagocytosis
2
Isolation and Flow Cytometry Analysis of Murine Epididymal Adipose Tissue
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Epididymal white adipose tissues were chopped with scissors for 4 minutes, followed by addition of 2 mL of collagenase II (SIGMA C6885, 400 U/mL) and incubation at 37 °C for 8 minutes. After digestion, single-cell suspensions were treated with 20 μL of 0.5 M EDTA and filtered using 70 μm meshes. The cells were centrifuged at 385 g for 10 minutes at 4 °C to remove floating adipocytes, and red blood cells were lysed in 1× RBC lysis buffer (eBioscience 00-4300-54) for 2 minutes on ice. After blocking of Fc receptors using TruStain FcX (Biolegend 101320), the cells were stained with the indicated fluorochrome conjugated antibodies. We used a LSRFortessa (BD) or FACSCanto II (BD) system, and analysed flow cytometric data using FlowJo (Tree Star Inc.). The antibodies used for flow cytometry were PE-conjugated anti-CD64 (FcγRI) (Biolegend, 139304), PE-conjugated anti-IgG2a,κ (Biolegend, 400507), PerCP-conjugated anti-CD45 (Biolegend, 103130), PE/cy7-conjugated anti-CD11c (Biolegend, 117318), APC-conjugated anti-CD11b (Biolegend, CD11b), and BV421-conjugated anti-SiglecF (BD Horizon, 562681).
3
Cell Surface Marker Evaluation
4
Multiparameter Flow Cytometry of Immune Cells
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PE-conjugated anti-CD23 (eBioscience-B3B4), FITC-conjugated anti-IgM (Sigma-μ chain specific) or brilliant violet (BV) 421-conjugated anti-IgM (BD pharmingen-R6-60.2), APC-conjugated anti-CD19 (BD bioscience-1D3), FITC-conjugated anti-CD3ε (Immunotools-145-2C11), APC-conjugated anti-CD11b (Biolegend- M1/70) and PE-conjugated anti-Gr-1(Immunotools- RB6-8C5) were used for flow cytometric analysis of blood leukocytes. Biotinylated anti-B220 (eBioscience-RA3-6B2) or Alexa 647-conjugated anti-B220 (BD Pharmingen- RA3-6B2), PE-conjugated anti-IgD (Biolegend-clone 11-26c.2a), FITC-conjugated anti-IgM (Sigma-μ chain specific) or BV 421-conjugated anti-IgM (BD Pharmingen- R6-60.2), FITC- conjugated anti-CD21 (BD Pharmingen- eBio8D9), PE-conjugated anti-CD23 (eBioscience-B3B4), APC-conjugated anti-CD19 (BD bioscience-1D3) or Alexa 700-conjugated anti-CD19 (BD Pharmingen-1D3) were used for flow cytometric analysis of BM or spleen cells. Biotinylated antibodies were detected with APC-conjugated streptavidin (Immunotools) or BV 605-conjugated streptavidin (BioLegend). Rat anti-mouse SIRPα (mAb P84; rat IgG1; a generous gift from Dr.Carl Lagenaur, university of Pittsburgh) was purified and conjugated to Alexa 488 as previously described [18 (link)].
5
Myeloid Differentiation of CD34+ HSPCs
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To differentiate HSPCs into myeloid lineage cells, a total of 1 × 104 CD34+ cells (in a volume of 1 mL) were seeded into each well of a 12-well plate (1 mL/well) and cultured in SFEM II supplemented with StemSpan Myeloid Expansion Supplement (STEMCELL Technologies, 02693). Every 2 days, 500 μL of fresh medium was added. Cells were collected by centrifugation (350 × g, 5 min) and resuspended in fresh medium every 7 days. Cells were analyzed by flow cytometry on days 7 and 14 for expression of myeloid lineage markers, using FITC-conjugated anti-CD14 (BioLegend, 325,603) and APC-conjugated anti-CD11b (BioLegend, 301,310) antibodies.
6
Mouse Kidney Cell Isolation and Immunophenotyping
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Mouse kidney tissue was diced and digested with Collagenase-I at 37°C for 1 hour. The suspension was filtered through a 40 um strainer to remove cell pellets and washed in PBS to generate a single cell suspension after lysis of red blood cells (BD, USA). The cells were washed twice with PBS. The third generation of hAD-MSCs were trypsinized and washed twice with PBS. The cells were then incubated with the following fluorescent antibodies and corresponding isotype controls (Cat.Number: 400605, 400611 and 400507, Biolegend, USA) for 30 minutes shielded from light at room temperature: FITC-conjugated anti-CD45, APC-conjugated anti-CD11b, PE-Cy7-conjugated anti-CD3, PE-conjugated anti-F4/80, APC-conjugated anti-CD34, PE-conjugated anti-CD31, FITC-conjugated anti-CD90, APC-conjugated anti-CD44 and PE-conjugated anti-105 (Biolegend, USA). The positive cells were sorted using a BD FACS and the data were analysed using the FlowJo v7.6.3 software.
7
Histological and Immunofluorescence Analysis of Liver
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For histology analysis, we imbedded the fixed livers in paraffin, then cut thin sections, and following standard procedures, stained them with hematoxylin and eosin. We quantified the liver histological score of inflammation according to Ishak inflammation score [43 (link), 44 (link)]. For immunofluorescence analysis, we cut frozen sections into 8μM slices in a cryostat microtome (Thermo) at -20°C and permeabilized and blocked them. We probed the tissues with appropriate, fluorescently labelled antibodies, including APC-conjugated anti-CD11b, PE-conjugated anti-CD68, and PE-conjugated anti-Ly6G (Biolegend). Finally, we dried the sections and observed them with a Nikon A1 confocal microscope.
8
Isolation and Culture of Endothelial Progenitor Cells
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Bone marrow cells were collected and cultured as aforementioned. Cell aliquots (1×106) were incubated for 20 min at 4°C with the following anti-mouse antibodies: APC-conjugated anti-CD11b (dilution, 1:100; BioLegend, Inc.), FITC-conjugated anti-CD31 (cat. no. 102506; dilution, 1:50; BioLegend, Inc.), Per CP-conjugated anti-CD144 (cat. no. 46-1441-82; dilution, 1:50; eBioscience; Thermo Fisher Scientific, Inc.) and PE-conjugated anti-CD133 (cat. no. 141203; dilution, 1:40; BioLegend, Inc.). Acquisition was performed using a FACSAria flow cytometer, and data were analyzed using FACSDiva software version 6.1.3. Sorted CD133+/CD31+/CD144+/CD11b− cells were enriched by further culture in endothelial growth basal medium (EBM)-2 (Lonza Group, Ltd., Basel, Switzerland).
9
Profiling Immune Cell Surface Markers
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The isolated bone marrow single cells were used for the detection of surface marker expressions, CD11b and Ly-6C, by flow cytometry. 200 μL immune cell resuspension was taken out of each sample and filtered by cell strainers. The cell resuspension was then stained for 30 min at 4 °C by APC-conjugated anti-CD11b and PE-conjugated anti-Ly-6C (BioLegend, San Diego, CA, USA). About 106 cells were recorded. Through the support of the laboratory, flow cytometry analysis could be performed by Beckman Coulter CytoFlex S, and the data were analyzed by CytExpert Software.
10
Comprehensive Lung Cell Immunophenotyping
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Single-lung-cell suspensions were blocked using anti-mouse CD16/CD32 (mouse BD Fc block, BD Biosciences, New Jersey, USA) at room temperature for 15 min prior to staining. Surface antigens were stained with the indicated conjugated antibodies at room temperature for 15 min. The following antibodies were used: APC-conjugated anti-CD4 (Thermo Fisher Scientific), PerCP-eFluor 710-conjugated anti-CD3e (Thermo Fisher Scientific), PE-Cyanine7-conjugated anti-CD45R (Thermo Fisher Scientific), APC/Cyanine7-conjugated anti-CD45 (BioLegend, San Diego, CA, USA), PE-conjugated anti-Siglec-F (Thermo Fisher Scientific), PerCD-eFluor710-conjugated anti-Ly6G, Alexa Fluor 700-conjugated anti-MHC Class II (I-A/I-E) (Thermo Fisher Scientific), eFluor 450-conjugated anti-F4/80 (Thermo Fisher Scientific), APC-conjugated anti-CD11b (BioLegend), FITC-conjugated anti-NK1.1 (BD Biosciences), PE-conjugated anti-CD49b (BioLegend), and PerCP-e710-conjugated anti-CD3e (BD Biosciences). For all experiments, cells were analyzed using a Gallios flow cytometer (Beckman Coulter). Panel setup and fluorescence compensation were performed using UltraCompeBeads™ Compensation Beads (Invitrogen). All analyses were performed using FlowJo software (Becton, Dickinson and Company, New Jersey, USA).
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