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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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, C3bi6,7,8,9, 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.......

Representative Results

The erythrocytes of three subjects whose density of CR1 is known ("low" subject [180 CR1/E], "medium" subject [646 CR1/E], and "high" subject [966 CR1/E]), and of two subjects whose CR1 density needed to be determined were immunostained by an anti-CR1 antibody coupled to an amplification system using the phycoerythrin fluorochrome. At the beginning, the CR1 density of the subjects from the low-high range was determined by the Scatchard method29 using radiolabeled antibodies.......

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 antibodies1,33. It is also possible to measure the con.......

Acknowledgements

We thank all the members of the URCACyt, flow cytometry technical platform, the staff of the Department of Immunology, and the staff of the Department of Internal Medicine and Geriatrics, who contributed to optimizing and validating the protocol. This work was funded by Reims University Hospitals (grant number AOL11UF9156).

....

Materials

NameCompanyCatalog NumberComments
1000E Barrier TipThermo Fischer Scientific , F-67403 Illkirch, France2079Esample pipetting
1-100 µL Bevelled, filter tipStarlab GmbH, D-22926 Ahrenburg, GermanyS1120-1840sample pipetting
Biotinylated anti-CR1 monoclonal antibody (J3D3)Home production of non-commercial monoclonal antibody, courtesy of Dr J. Cookimmunostaining
Blouseprotection
Bovin serum albumin (7,5%)Thermo Fischer Scientific , F-67403 Illkirch, France15260037cytometry
CentrifugeThermo Fischer Scientific , F-67403 Illkirch, France11176917centrifugation
Clean SolutionBD, F-38801 Le Pont de Claix, France340345cytometry
Comorack-96Dominique DUTSCHER SAS, F-67172 Brumath944060Prack
Cytometer Setup & Tracking Beads KitBD, F-38801 Le Pont de Claix, France655051cytometry
Formaldehyde solution 36.5 %Sigma Aldrich, F-38070 Saint Quentin Fallavier, FranceF8775-25MLFixation
10 µL Graduated, filter tipStarlab GmbH, D-22926 Ahrenburg, GermanyS1121-3810sample pipetting
LSRFORTESSA Flow CytometerBD, F-38801 Le Pont de Claix, France647788cytometry
Microman Capillary PistonsDominique DUTSCHER SAS, F-67172 Brumath067494sample pipetting
Micronic 1.40 mL round bottom tubesDominique DUTSCHER SAS, F-67172 BrumathMP32051mix
Micropipette Microman - type M25 -Dominique DUTSCHER SAS, F-67172 Brumath066379sample pipetting
Phosphate buffered Saline (PBS)Thermo Fischer Scientific , F-67403 Illkirch, France10010031cytometry
Pipette PS 325 mm, 10 mLDominique DUTSCHER SAS, F-67172 Brumath391952sample pipetting
powder-free Nitrile Exam glovesMedline Industries, Inc, Mundelein, IL 60060, USA486802sample protection
Reference 2 pipette, 0,5-10 µLEppendorf France SAS, F-78360 Montesson, France4920000024sample pipetting
Reference 2 pipette, 20-100 µLEppendorf France SAS, F-78360 Montesson, France4920000059sample pipetting
Reference 2 pipette, 100-1000 µLEppendorf France SAS, F-78360 Montesson, France4920000083sample pipetting
Rinse SolutionBD, F-38801 Le Pont de Claix, France340346cytometry
Round bottom tubeSarstedt, F-70150 Marnay, France55.1579cytometry
Safe-Lock Tubes, 1.5 mLEppendorf France SAS, F-78360 Montesson, France0030120086mix
streptavidin R-PETebu Bio, F-78612 Le Perray-en-Yvelines, FranceAS-60669immunostaining
Tapered Centrifuge Tubes 50 mLThermo Fischer Scientific , F-67403 Illkirch, France10203001mix
Vector anti streptavidin biotinEurobio Ingen, F-91953 Les Ulis, FranceBA-0500immunostaining
Vortex-Genie 2Scientific Industries, Inc, Bohemia, NY 111716, USASI-0236mix

References

  1. Fearon, D. T. Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte. Journal of Experimental Medicine. 152 (1), 20-30 (1980).
  2. Ross, G. D., Winchester, R. J., Rabellino, E. M., Hoffman, T.

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