Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

Summary

The aim of this method is to determine the CR1 density in the erythrocytes of any subject by comparing with three subjects whose erythrocyte CR1 density is known. The method uses flow cytometry after immunostaining of the subjects' erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

Abstract

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100–1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer's disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

Introduction

CR1 (complement receptor type 1, CD35) is a 200 kDa transmembrane glycoprotein present on the surface of many cell types, such as erythrocytes¹, B lymphocytes², monocytic cells, some T cells, follicular dendritic cells³, fetal astrocytes⁴, and glomerular podocytes⁵. CR1 interfering with its ligands C3b, C4b, C3bi^6,7,8,9, a subunit of the first complement component, C1q¹⁰ and MBL (mannan-binding lecti....

Protocol

The protocol for human blood collection and handling was reviewed and approved by the regional ethics committee (CPP Est II), and the protocol number is 2011-A00594-37. Because the following protocol describes the handling of human blood, institutional guidelines for disposing of biohazardous material should be followed. Laboratory safety equipment, such as lab coats and gloves, should be worn.

1. Erythrocyte washing

NOTE: The day before handling, prepare a PBS-BSA bu.......

Discussion

Several techniques are available to determine the density of erythrocyte CR1 (CR1/E). The first techniques used were the agglutination of red blood cells by anti-CR1 antibodies³¹ and the formation of rosettes in the presence of erythrocytes coated with C3b³². These rudimentary techniques were rapidly replaced by immunostaining methods using radiolabeled anti-CR1 antibodies^1,33. It is also possible to measure the con.......

Materials

Name	Company	Catalog Number	Comments
1000E Barrier Tip	Thermo Fischer Scientific , F-67403 Illkirch, France	2079E	sample pipetting
1-100 µL Bevelled, filter tip	Starlab GmbH, D-22926 Ahrenburg, Germany	S1120-1840	sample pipetting
Biotinylated anti-CR1 monoclonal antibody (J3D3)	Home production of non-commercial monoclonal antibody, courtesy of Dr J. Cook		immunostaining
Blouse			protection
Bovin serum albumin (7,5%)	Thermo Fischer Scientific , F-67403 Illkirch, France	15260037	cytometry
Centrifuge	Thermo Fischer Scientific , F-67403 Illkirch, France	11176917	centrifugation
Clean Solution	BD, F-38801 Le Pont de Claix, France	340345	cytometry
Comorack-96	Dominique DUTSCHER SAS, F-67172 Brumath	944060P	rack
Cytometer Setup & Tracking Beads Kit	BD, F-38801 Le Pont de Claix, France	655051	cytometry
Formaldehyde solution 36.5 %	Sigma Aldrich, F-38070 Saint Quentin Fallavier, France	F8775-25ML	Fixation
10 µL Graduated, filter tip	Starlab GmbH, D-22926 Ahrenburg, Germany	S1121-3810	sample pipetting
LSRFORTESSA Flow Cytometer	BD, F-38801 Le Pont de Claix, France	647788	cytometry
Microman Capillary Pistons	Dominique DUTSCHER SAS, F-67172 Brumath	067494	sample pipetting
Micronic 1.40 mL round bottom tubes	Dominique DUTSCHER SAS, F-67172 Brumath	MP32051	mix
Micropipette Microman - type M25 -	Dominique DUTSCHER SAS, F-67172 Brumath	066379	sample pipetting
Phosphate buffered Saline (PBS)	Thermo Fischer Scientific , F-67403 Illkirch, France	10010031	cytometry
Pipette PS 325 mm, 10 mL	Dominique DUTSCHER SAS, F-67172 Brumath	391952	sample pipetting
powder-free Nitrile Exam gloves	Medline Industries, Inc, Mundelein, IL 60060, USA	486802	sample protection
Reference 2 pipette, 0,5-10 µL	Eppendorf France SAS, F-78360 Montesson, France	4920000024	sample pipetting
Reference 2 pipette, 20-100 µL	Eppendorf France SAS, F-78360 Montesson, France	4920000059	sample pipetting
Reference 2 pipette, 100-1000 µL	Eppendorf France SAS, F-78360 Montesson, France	4920000083	sample pipetting
Rinse Solution	BD, F-38801 Le Pont de Claix, France	340346	cytometry
Round bottom tube	Sarstedt, F-70150 Marnay, France	55.1579	cytometry
Safe-Lock Tubes, 1.5 mL	Eppendorf France SAS, F-78360 Montesson, France	0030120086	mix
streptavidin R-PE	Tebu Bio, F-78612 Le Perray-en-Yvelines, France	AS-60669	immunostaining
Tapered Centrifuge Tubes 50 mL	Thermo Fischer Scientific , F-67403 Illkirch, France	10203001	mix
Vector anti streptavidin biotin	Eurobio Ingen, F-91953 Les Ulis, France	BA-0500	immunostaining
Vortex-Genie 2	Scientific Industries, Inc, Bohemia, NY 111716, USA	SI-0236	mix