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Species-specific binding of soluble human SIRPα to RBCs and CD47-coated beads. (A) Fresh human and sheep RBCs binding to soluble hSIRPα (4 μM of GST conjugate), as detected by <t>FITC-anti-GST.</t> “Bkgd” is obtained with RBCs plus antibody. (B) Affinity of hCD47-coated beads binding to soluble hSIRPα based on flow cytometry (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200708043/DC1 ). Saturation binding fit gave the indicated dissociation constant, K d . Because this is a 3D binding constant relevant to binding in a narrow membrane gap between two cells, it is equivalent to K d ≈ <t>1</t> <t>molecule/[10</t> nm x (10 μm) 2 ], which is the concentration of free SIRPα that would half-saturate CD47 on a surface. The inset shows inhibition of soluble hSIRPα binding to hCD47 beads by using anti-CD47 F(ab′) 2 generated from B6H12 antibody; similar inhibition is obtained with human RBCs.
Quantum Fitc Molecule Of Equivalent Soluble Fluorochrome (Mesf) Units, supplied by Bangs Laboratories, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Species-specific binding of soluble human SIRPα to RBCs and CD47-coated beads. (A) Fresh human and sheep RBCs binding to soluble hSIRPα (4 μM of GST conjugate), as detected by <t>FITC-anti-GST.</t> “Bkgd” is obtained with RBCs plus antibody. (B) Affinity of hCD47-coated beads binding to soluble hSIRPα based on flow cytometry (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200708043/DC1 ). Saturation binding fit gave the indicated dissociation constant, K d . Because this is a 3D binding constant relevant to binding in a narrow membrane gap between two cells, it is equivalent to K d ≈ <t>1</t> <t>molecule/[10</t> nm x (10 μm) 2 ], which is the concentration of free SIRPα that would half-saturate CD47 on a surface. The inset shows inhibition of soluble hSIRPα binding to hCD47 beads by using anti-CD47 F(ab′) 2 generated from B6H12 antibody; similar inhibition is obtained with human RBCs.
Quantum Mesf Fitc Kit, supplied by Bangs Laboratories, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Species-specific binding of soluble human SIRPα to RBCs and CD47-coated beads. (A) Fresh human and sheep RBCs binding to soluble hSIRPα (4 μM of GST conjugate), as detected by <t>FITC-anti-GST.</t> “Bkgd” is obtained with RBCs plus antibody. (B) Affinity of hCD47-coated beads binding to soluble hSIRPα based on flow cytometry (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200708043/DC1 ). Saturation binding fit gave the indicated dissociation constant, K d . Because this is a 3D binding constant relevant to binding in a narrow membrane gap between two cells, it is equivalent to K d ≈ <t>1</t> <t>molecule/[10</t> nm x (10 μm) 2 ], which is the concentration of free SIRPα that would half-saturate CD47 on a surface. The inset shows inhibition of soluble hSIRPα binding to hCD47 beads by using anti-CD47 F(ab′) 2 generated from B6H12 antibody; similar inhibition is obtained with human RBCs.
Fitc Mesf Beads, supplied by Bangs Laboratories, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Species-specific binding of soluble human SIRPα to RBCs and CD47-coated beads. (A) Fresh human and sheep RBCs binding to soluble hSIRPα (4 μM of GST conjugate), as detected by <t>FITC-anti-GST.</t> “Bkgd” is obtained with RBCs plus antibody. (B) Affinity of hCD47-coated beads binding to soluble hSIRPα based on flow cytometry (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200708043/DC1 ). Saturation binding fit gave the indicated dissociation constant, K d . Because this is a 3D binding constant relevant to binding in a narrow membrane gap between two cells, it is equivalent to K d ≈ <t>1</t> <t>molecule/[10</t> nm x (10 μm) 2 ], which is the concentration of free SIRPα that would half-saturate CD47 on a surface. The inset shows inhibition of soluble hSIRPα binding to hCD47 beads by using anti-CD47 F(ab′) 2 generated from B6H12 antibody; similar inhibition is obtained with human RBCs.
Designated Molecules Of Equivalent Soluble Fluorochrome (Mesf) Beads Quantum Apc Mesf Cat. No. 823a, supplied by Bangs Laboratories, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Species-specific binding of soluble human SIRPα to RBCs and CD47-coated beads. (A) Fresh human and sheep RBCs binding to soluble hSIRPα (4 μM of GST conjugate), as detected by <t>FITC-anti-GST.</t> “Bkgd” is obtained with RBCs plus antibody. (B) Affinity of hCD47-coated beads binding to soluble hSIRPα based on flow cytometry (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200708043/DC1 ). Saturation binding fit gave the indicated dissociation constant, K d . Because this is a 3D binding constant relevant to binding in a narrow membrane gap between two cells, it is equivalent to K d ≈ <t>1</t> <t>molecule/[10</t> nm x (10 μm) 2 ], which is the concentration of free SIRPα that would half-saturate CD47 on a surface. The inset shows inhibition of soluble hSIRPα binding to hCD47 beads by using anti-CD47 F(ab′) 2 generated from B6H12 antibody; similar inhibition is obtained with human RBCs.
Quantum Fitc Mesf, supplied by Quantum Dot Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Species-specific binding of soluble human SIRPα to RBCs and CD47-coated beads. (A) Fresh human and sheep RBCs binding to soluble hSIRPα (4 μM of GST conjugate), as detected by <t>FITC-anti-GST.</t> “Bkgd” is obtained with RBCs plus antibody. (B) Affinity of hCD47-coated beads binding to soluble hSIRPα based on flow cytometry (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200708043/DC1 ). Saturation binding fit gave the indicated dissociation constant, K d . Because this is a 3D binding constant relevant to binding in a narrow membrane gap between two cells, it is equivalent to K d ≈ <t>1</t> <t>molecule/[10</t> nm x (10 μm) 2 ], which is the concentration of free SIRPα that would half-saturate CD47 on a surface. The inset shows inhibition of soluble hSIRPα binding to hCD47 beads by using anti-CD47 F(ab′) 2 generated from B6H12 antibody; similar inhibition is obtained with human RBCs.
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Species-specific binding of soluble human SIRPα to RBCs and CD47-coated beads. (A) Fresh human and sheep RBCs binding to soluble hSIRPα (4 μM of GST conjugate), as detected by <t>FITC-anti-GST.</t> “Bkgd” is obtained with RBCs plus antibody. (B) Affinity of hCD47-coated beads binding to soluble hSIRPα based on flow cytometry (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200708043/DC1 ). Saturation binding fit gave the indicated dissociation constant, K d . Because this is a 3D binding constant relevant to binding in a narrow membrane gap between two cells, it is equivalent to K d ≈ <t>1</t> <t>molecule/[10</t> nm x (10 μm) 2 ], which is the concentration of free SIRPα that would half-saturate CD47 on a surface. The inset shows inhibition of soluble hSIRPα binding to hCD47 beads by using anti-CD47 F(ab′) 2 generated from B6H12 antibody; similar inhibition is obtained with human RBCs.
Quantum Fitc Mesf Calibration Beads (Bangs Labs, Cat. No. 825b), supplied by Bangs Laboratories, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Species-specific binding of soluble human SIRPα to RBCs and CD47-coated beads. (A) Fresh human and sheep RBCs binding to soluble hSIRPα (4 μM of GST conjugate), as detected by <t>FITC-anti-GST.</t> “Bkgd” is obtained with RBCs plus antibody. (B) Affinity of hCD47-coated beads binding to soluble hSIRPα based on flow cytometry (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200708043/DC1 ). Saturation binding fit gave the indicated dissociation constant, K d . Because this is a 3D binding constant relevant to binding in a narrow membrane gap between two cells, it is equivalent to K d ≈ <t>1</t> <t>molecule/[10</t> nm x (10 μm) 2 ], which is the concentration of free SIRPα that would half-saturate CD47 on a surface. The inset shows inhibition of soluble hSIRPα binding to hCD47 beads by using anti-CD47 F(ab′) 2 generated from B6H12 antibody; similar inhibition is obtained with human RBCs.
Mesf Beads, supplied by Bangs Laboratories, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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QUANTUM FITC-5 MESF; QUANTUM™ FITC-5 MESF
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Species-specific binding of soluble human SIRPα to RBCs and CD47-coated beads. (A) Fresh human and sheep RBCs binding to soluble hSIRPα (4 μM of GST conjugate), as detected by FITC-anti-GST. “Bkgd” is obtained with RBCs plus antibody. (B) Affinity of hCD47-coated beads binding to soluble hSIRPα based on flow cytometry (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200708043/DC1 ). Saturation binding fit gave the indicated dissociation constant, K d . Because this is a 3D binding constant relevant to binding in a narrow membrane gap between two cells, it is equivalent to K d ≈ 1 molecule/[10 nm x (10 μm) 2 ], which is the concentration of free SIRPα that would half-saturate CD47 on a surface. The inset shows inhibition of soluble hSIRPα binding to hCD47 beads by using anti-CD47 F(ab′) 2 generated from B6H12 antibody; similar inhibition is obtained with human RBCs.

Journal: The Journal of Cell Biology

Article Title: Inhibition of “self” engulfment through deactivation of myosin-II at the phagocytic synapse between human cells

doi: 10.1083/jcb.200708043

Figure Lengend Snippet: Species-specific binding of soluble human SIRPα to RBCs and CD47-coated beads. (A) Fresh human and sheep RBCs binding to soluble hSIRPα (4 μM of GST conjugate), as detected by FITC-anti-GST. “Bkgd” is obtained with RBCs plus antibody. (B) Affinity of hCD47-coated beads binding to soluble hSIRPα based on flow cytometry (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200708043/DC1 ). Saturation binding fit gave the indicated dissociation constant, K d . Because this is a 3D binding constant relevant to binding in a narrow membrane gap between two cells, it is equivalent to K d ≈ 1 molecule/[10 nm x (10 μm) 2 ], which is the concentration of free SIRPα that would half-saturate CD47 on a surface. The inset shows inhibition of soluble hSIRPα binding to hCD47 beads by using anti-CD47 F(ab′) 2 generated from B6H12 antibody; similar inhibition is obtained with human RBCs.

Article Snippet: The fluorescent intensities were standardized using Quantum FITC Molecule of Equivalent Soluble Fluorochrome (MESF) units (Bangs Laboratories).

Techniques: Binding Assay, Flow Cytometry, Membrane, Concentration Assay, Inhibition, Generated