Purified anti-CD47 mAbs clones CC2C6 and 2D3 (BioLegend) and clone B6H12 (BD Biosciences) and purified anti-SIRPα mAbs (clone SE5A5) and G1 isotype mAbs were used at 10 μg/ml (BioLegend). Anti–Mac-1 was reconstituted by mixing anti-CD11b (clone ICRF44) and CD18 (clone TS1/18) mAbs at a final concentration of 10 μg/ml for each (BioLegend). Anti-CD3 mAbs (clone UCHT1) were prepared in the laboratory and used at 10 μg/ml. Recombinant SIRPα was prepared as in (30 (link)) and was used as a monomer or multimerized with Neutravidin at a saturating concentration of 5 μM. RGDS and LGDP were used, respectively, at 40 μg/ml and 100 μg/ml (Sigma Aldrich Merck). 4N1K, a CD47-binding domain adhesive peptide derived from TPS1, was used at 10 μM (Eurogentec). CC2C6-F(ab)’2 was prepared using F(ab’)2 Preparation Kit (Pierce) as described by the manufacturer using an optimized 1-h digestion time at 37°C. Digestion was verified by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and CD47 binding activity was measured against the parent CC2C6 using AlphaScreen as described in (30 (link)).
Anti cd11b clone icrf44
Manufactured by BioLegend
Anti-CD11b (clone ICRF44) is a monoclonal antibody that binds to the CD11b antigen. CD11b is a cell surface receptor expressed on various immune cells, including monocytes, macrophages, and neutrophils. This antibody can be used for the identification and characterization of cells expressing CD11b.
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Lab products found in correlation
Cd18 clone ts1 18, by BioLegend (1 mentions)
Lsrfortessa x 20 cell analyzer, by BD (1 mentions)
Anti cd86 clone it2, by BioLegend (1 mentions)
Mouse igg1, by BioLegend (1 mentions)
Mouse igg2bκ, by BioLegend (1 mentions)
Anti cd14 clone m5e2, by BioLegend (1 mentions)
Fluoview 500 laser scanning confocal microscope, by Olympus (1 mentions)
Phalloidin alexa488, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti cd11b clone icrf44
1
Anti-CD47 and Anti-SIRPα Antibodies Protocol
2
Comprehensive Immune Phenotyping of Cells
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Cells were washed 2 times with cold PBS, scraped and collected by centrifugation and resuspended in FACS buffer (PBS supplemented with 0,5% bovine serum albumin and 5mM of EDTA). Cells were stained with anti-CD11b (clone ICRF44, FITC conjugated; Biolegend) or isotype control (mouse IgG1 FITC conjugated biolegend);, anti-CD14 (clone M5e2, APC conjugated; Biolegend) or isotype control (mouse, IgG2a, κ, APC conjugated, Biolegend), anti-CD206 (clone 15-2, APC/cy7 conjugated; Biolegend) or isotype control (mouse IgG1, APC/cy7 conjugated, Biolegend), anti-CD86 (clone IT2.2, Alexa Fluor 488 conjugated; Biolegend) or isotype control (mouse IgG2b, κ, Alexa Fluor 488 conjugated, Biolegend) and anti-CD163 (clone RM3/1, Alexa Fluor 647 conjugated; Biolegend) or isotype control (mouse IgG1, κ, Alexa Fluor 647 conjugated, Biolegend) Cells were analyzed on a BD LSRFortessa X-20 Cell Analyzer (BD Bioscience) with post-processing in FlowJo software (Tree star Inc). Cell populations were gated on forward and side scatter to select intact single cells. The gating strategy and a representative flow diagram is shown in S2 Fig .
3
Neutrophil Adhesion and Spreading Assay
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Lab-Tek borosilicate chambered coverglass microplates (NUNC, Rochester, NY) were coated with 50 μg/ml FH or BSA in modified Hank's buffer overnight, then washed three times.
Neutrophils (2 × 10 5 cells) in 200 µl were added and allowed to adhere/spread for 60 min at 37°C in CO2 thermostat, then fixed with 2% paraformaldehyde for 5 min at 37 o C, followed by washing twice with PBS. For blocking experiments, cells were preincubated with 50 µg/ml anti-CD11b (clone: ICRF44; Biolegend, San Diego, CA) or with control mouse IgG1 mAb (in house) for 20 min at 4°C. The adhered cells were stained with Phalloidin Alexa-488 (Molecular Probes-Invitrogen; 1:100 in 0.1% Triton X-100) for 5 min at 37°C, and then washed four times with PBS.
The contact surface of the cells was monitored in Olympus FluoView 500 laser-scanning confocal microscope (excitation: 488 nm). For measuring of the contact zone area we used ImageJ software (http://rsbweb.nih.gov/ij) with Analyze Particle tool.
Neutrophils (2 × 10 5 cells) in 200 µl were added and allowed to adhere/spread for 60 min at 37°C in CO2 thermostat, then fixed with 2% paraformaldehyde for 5 min at 37 o C, followed by washing twice with PBS. For blocking experiments, cells were preincubated with 50 µg/ml anti-CD11b (clone: ICRF44; Biolegend, San Diego, CA) or with control mouse IgG1 mAb (in house) for 20 min at 4°C. The adhered cells were stained with Phalloidin Alexa-488 (Molecular Probes-Invitrogen; 1:100 in 0.1% Triton X-100) for 5 min at 37°C, and then washed four times with PBS.
The contact surface of the cells was monitored in Olympus FluoView 500 laser-scanning confocal microscope (excitation: 488 nm). For measuring of the contact zone area we used ImageJ software (http://rsbweb.nih.gov/ij) with Analyze Particle tool.
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