Name: measuring erythrocyte complement receptor 1 using flow cytometry
Uploaded: 2020-05-19T12:30:00
Description: Watch this Scientific Journal Video about Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry at JoVE.com

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).

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...

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...

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 lectin)11 inhibits the activation of complement and is involved in humoral and cellular immune response.
In primates, including humans, erythrocyte CR1 is involved in the transport of immune complexes to the liver and spleen, to purify the blood and prevent their accumulation in vulnerable tissues such as the skin or kidneys12,13,14. This phenomenon of immune adhesion between immune complexes and erythrocytes depends on the number of CR1 molecules15. In humans, the mean density of CR1/E is only 500 (i.e., 500 molecules of CR1 per erythrocyte). This density varies from one individual to another (100&#8211;1,200 CR1/E) and from one erythrocyte to another in the same individual. Some individuals of &#34;null&#34; phenotype express fewer than 20 CR1/E16.
The density of CR1/E is regulated by two co-dominant autosomal alleles linked to a point mutation in intron 27 of the gene coding for CR1*117,18. This mutation produces an additional restriction site for the HindIII enzyme. The restriction fragments obtained after digestion with HindIII in this case are 7.4 kb for the allele linked to a strong expression of CR1 (H: high allele) and 6.9 kb for the allele linked to low CR1 expression (L: low allele). This link is found in Caucasians and Asians but not in people of African descent19.
The level of expression of erythrocyte CR1 is also correlated with the presence of point nucleotide mutations in exon 13 encoding SCR 10 (I643T) and in exon 19 encoding SCR16 (Q981H). It is high in homozygous 643I/981Q and low in homozygous 643T/981H individuals20. Thus, &#34;low&#34; individuals express around 150 CR1/E, &#34;medium&#34; individuals express around 500 CR1/E, and &#34;high&#34; individuals express around 1,000 CR1/E.
In addition to this erythrocyte density polymorphism, CR1 is characterized by a length polymorphism corresponding to four allotypes of different sizes: CR1*1 (190 kDa), CR1*2 (220 kDa), CR1*3 (160 kDa), and CR1*4 (250 kDa)21 and an antigenic polymorphism corresponding to the blood group KN22.
We present our method based on flow cytometry to determine the density of CR1/E. Using three subjects whose CR1/E density is known, expressing a low density level (180 CR1/E), a medium density level (646 CR1/E), and a high density level (966 CR1/E), it is easy to measure the mean fluorescence intensity (MFI) of their erythrocytes or red blood cells (RBC), or RBC MFI, after anti-CR1 immunostaining using a flow cytometer. One can then plot a standard line representing the MFI as a function of CR1/E density. Measuring the MFI of subjects whose CR1/E density is not known and comparing it to this standard line, it is possible to determine the individuals&#39; CR1/E density. This technique has been used for many years in the laboratory, and has enabled us to detect a reduction in the expression of erythrocyte CR1 in many pathologies such as systemic lupus erythematosus (SLE)23, Acquired immunodeficiency syndrome (AIDS)24, malaria25, and recently Alzheimer&#39;s disease (AD)26,27. The development of drugs targeting CR1 to couple with erythrocytes, as in the case of anti-thrombotic drugs28 requires the evaluation of CR1/E density, and the availability of a robust technique to quantify CR1.
The protocol presented runs in singlicate. It is adaptable to determine the density of CR1/E on many individuals using specific commercially available 96 well plates (see Table of Materials). To this end, it is easy to adapt our method to any 96 well plate. For each sample, a cell suspension of erythrocytes (0.5 x 106&#8211;1 x 106 erythrocytes) is distributed per well. For each well, first the primary anti-CR1 antibody is added, then streptavidin PE, the secondary anti-streptavidin antibody, and again streptavidin PE, using the same dilutions as those of our method, but by adapting volumes and respecting proportionality.
The blood samples from subjects of the range and from subjects to be quantified for CR1 should be drawn at the same time, stored in the refrigerator at 4 &#176;C, and handled at 4 &#176;C (on ice and/or in the refrigerator).

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			<td height="42" style="height:42px;width:216px;">1000E Barrier Tip</td>
			<td style="width:239px;">Thermo Fischer Scientific , F-67403 Illkirch, France</td>
			<td style="width:119px;">2079E</td>
			<td style="width:179px;">sample pipetting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1-100 &#181;L Bevelled, filter tip</td>
			<td style="width:239px;">Starlab GmbH, D-22926 Ahrenburg, Germany</td>
			<td style="width:119px;">S1120-1840</td>
			<td>sample pipetting</td>
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			<td height="63" style="height:63px;width:216px;">Biotinylated anti-CR1 monoclonal antibody (J3D3)</td>
			<td style="width:239px;">Home production of non-commercial monoclonal antibody, courtesy of Dr J. Cook</td>
			<td style="width:119px;"></td>
			<td>immunostaining</td>
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			<td height="21" style="height:21px;width:216px;">Blouse</td>
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			<td style="width:119px;"></td>
			<td>protection</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bovin serum albumin (7,5%)</td>
			<td style="width:239px;">Thermo Fischer Scientific , F-67403 Illkirch, France</td>
			<td style="width:119px;">15260037</td>
			<td>cytometry</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge</td>
			<td style="width:239px;">Thermo Fischer Scientific , F-67403 Illkirch, France</td>
			<td style="width:119px;">11176917</td>
			<td>centrifugation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Clean Solution</td>
			<td style="width:239px;">BD, F-38801 Le Pont de Claix, France</td>
			<td style="width:119px;">340345</td>
			<td>cytometry</td>
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			<td height="42" style="height:42px;">Comorack-96</td>
			<td style="width:239px;">Dominique DUTSCHER SAS, F-67172 Brumath</td>
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			<td>rack</td>
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			<td height="48" style="height:48px;width:216px;">Cytometer Setup &#38; Tracking Beads Kit</td>
			<td style="width:239px;">BD, F-38801 Le Pont de Claix, France</td>
			<td style="width:119px;">655051</td>
			<td>cytometry</td>
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			<td height="48" style="height:48px;width:216px;">Formaldehyde solution 36.5 %</td>
			<td style="width:239px;">Sigma Aldrich, F-38070 Saint Quentin Fallavier, France</td>
			<td style="width:119px;">F8775-25ML</td>
			<td>Fixation</td>
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		<tr height="48">
			<td height="48" style="height:48px;width:216px;">10 &#181;L Graduated, filter tip</td>
			<td style="width:239px;">Starlab GmbH, D-22926 Ahrenburg, Germany</td>
			<td style="width:119px;">S1121-3810</td>
			<td>sample pipetting</td>
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		<tr height="48">
			<td height="48" style="height:48px;width:216px;">LSRFORTESSA Flow Cytometer</td>
			<td style="width:239px;">BD, F-38801 Le Pont de Claix, France</td>
			<td style="width:119px;">647788</td>
			<td>cytometry</td>
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			<td height="48" style="height:48px;width:216px;">Microman Capillary Pistons</td>
			<td style="width:239px;">Dominique DUTSCHER SAS, F-67172 Brumath</td>
			<td style="width:119px;">067494</td>
			<td>sample pipetting</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:216px;">Micronic 1.40 mL round bottom tubes</td>
			<td style="width:239px;">Dominique DUTSCHER SAS, F-67172 Brumath</td>
			<td style="width:119px;">MP32051</td>
			<td>mix</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:216px;">Micropipette Microman - type M25 -</td>
			<td style="width:239px;">Dominique DUTSCHER SAS, F-67172 Brumath</td>
			<td style="width:119px;">066379</td>
			<td>sample pipetting</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:216px;">Phosphate buffered Saline (PBS)</td>
			<td style="width:239px;">Thermo Fischer Scientific , F-67403 Illkirch, France</td>
			<td style="width:119px;">10010031</td>
			<td>cytometry</td>
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		<tr height="48">
			<td height="48" style="height:48px;width:216px;">Pipette PS 325 mm, 10 mL</td>
			<td style="width:239px;">Dominique DUTSCHER SAS, F-67172 Brumath</td>
			<td style="width:119px;">391952</td>
			<td>sample pipetting</td>
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			<td height="48" style="height:48px;width:216px;">powder-free Nitrile Exam gloves</td>
			<td style="width:239px;">Medline Industries, Inc, Mundelein, IL 60060, USA</td>
			<td style="width:119px;">486802</td>
			<td>sample protection</td>
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			<td height="48" style="height:48px;width:216px;">Reference 2 pipette, 0,5-10 &#181;L</td>
			<td style="width:239px;">Eppendorf France SAS, F-78360 Montesson, France</td>
			<td style="width:119px;">4920000024</td>
			<td>sample pipetting</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:216px;">Reference 2 pipette, 20-100 &#181;L</td>
			<td style="width:239px;">Eppendorf France SAS, F-78360 Montesson, France</td>
			<td style="width:119px;">4920000059</td>
			<td>sample pipetting</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Reference 2 pipette, 100-1000 &#181;L</td>
			<td style="width:239px;">Eppendorf France SAS, F-78360 Montesson, France</td>
			<td style="width:119px;">4920000083</td>
			<td>sample pipetting</td>
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		<tr height="55">
			<td height="55" style="height:55px;">Rinse Solution</td>
			<td style="width:239px;">BD, F-38801 Le Pont de Claix, France</td>
			<td style="width:119px;">340346</td>
			<td>cytometry</td>
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		<tr height="55">
			<td height="55" style="height:55px;">Round bottom tube</td>
			<td style="width:239px;">Sarstedt, F-70150 Marnay, France</td>
			<td style="width:119px;">55.1579</td>
			<td>cytometry</td>
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			<td height="48" style="height:48px;width:216px;">Safe-Lock Tubes, 1.5 mL</td>
			<td style="width:239px;">Eppendorf France SAS, F-78360 Montesson, France</td>
			<td style="width:119px;">0030120086</td>
			<td>mix</td>
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		<tr height="48">
			<td height="48" style="height:48px;width:216px;">streptavidin R-PE</td>
			<td style="width:239px;">Tebu Bio, F-78612 Le Perray-en-Yvelines, France</td>
			<td style="width:119px;">AS-60669</td>
			<td>immunostaining</td>
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		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Tapered Centrifuge Tubes 50 mL</td>
			<td style="width:239px;">Thermo Fischer Scientific , F-67403 Illkirch, France</td>
			<td>10203001</td>
			<td>mix</td>
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			<td height="42" style="height:42px;width:216px;">Vector anti streptavidin biotin</td>
			<td style="width:239px;">Eurobio Ingen, F-91953 Les Ulis, France</td>
			<td style="width:119px;">BA-0500</td>
			<td>immunostaining</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vortex-Genie 2</td>
			<td style="width:239px;">Scientific Industries, Inc, Bohemia, NY 111716, USA</td>
			<td style="width:119px;">SI-0236</td>
			<td>mix</td>
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measuring erythrocyte complement receptor 1 using flow cytometry

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&#8211;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&#39;s disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

The erythrocytes of three subjects whose density of CR1 is known (&#34;low&#34; subject [180 CR1/E], &#34;medium&#34; subject [646 CR1/E], and &#34;high&#34; 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...

Watch this Scientific Journal Video about Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry at JoVE.com

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

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&#39; erythrocytes by an anti-CR1 monoclonal antibody coupled to an amplified system using phycoerythrin (PE).

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...

Research

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