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 buffer with phosphate buffered saline (PBS) containing 0.15% of bovine serum albumin (BSA) and place it in the refrigerator at 4 &#176;C. This buffer will be used as a washing buffer and as a dilution buffer.
<ol>
	<li>Pipette 20 mL of PBS-BSA into a 50 mL tube.</li>
	<li>Aspirate 250 &#181;L of sodium ethylenediamine tetraacetic acid (EDTA) anticoagulated whole blood from blood storage tubes and add to the tube containing the 20 mL of PBS-BSA. Close the tube by screwing the cap. Mix gently by inverting the tube 2x.</li>
	<li>Centrifuge the tube for 10 min at 4 &#176;C at 430 x g. Remove and discard the supernatant using a 10 mL pipette. Resuspend the pellet in the residual volume of supernatant by gentle and careful pipetting.</li>
	<li>Add 20 mL of cold PBS-BSA (4 &#176;C) into the tube containing the pellet. Centrifuge the tube for 10 min at 4 &#176;C at 430 x g. Remove and discard the supernatant using a 10 mL pipette.</li>
	<li>Add 20 mL of cold PBS-BSA (4 &#176;C) into the tube containing the pellet. Centrifuge the tube for 10 min at 4 &#176;C at 430 x g. Leave the tube in the centrifuge at 4 &#176;C and go to section 2.</li>
</ol>
2. Erythrocyte dilution
<ol>
	<li>Pipette 3 mL of cold PBS-BSA into a 50 mL tube and store it at 4 &#176;C on a rack in ice.</li>
	<li>Put the centrifuged tube containing the erythrocytes (step 1.4) on a rack placed in ice.</li>
	<li>Pipette 8 &#181;L of pelleted erythrocytes using the pipette and add to the 50 mL tube containing the 3 mL of PBS-BSA to obtain the erythrocyte dilution. Mix the tube gently by hand to obtain a homogeneous cell suspension of erythrocytes.</li>
</ol>
3. Erythrocyte immunostaining
<ol>
	<li>Pipette 100 &#181;L of erythrocyte dilution (obtained in section 4) and add to 1.4 mL tubes.</li>
	<li>Centrifuge the tubes for 5 min at 4 &#176;C at 430 x g. During centrifugation, prepare a dilution of biotinylated anti-CR1 J3D3 antibody at a concentration of 0.05 &#956;g/&#956;L in PBS-BSA buffer.</li>
	<li>Once the centrifugation is done, remove and discard the supernatant.</li>
	<li>Add 20 &#181;L of biotinylated anti-CR1 J3D3 directly to the pellet. To prepare the negative control, add 20 &#181;L of PBS-BSA buffer instead. Mix the tubes gently and incubate for 45 min at 4 &#176;C.</li>
	<li>After 45 min of incubation, add 750 &#181;L of PBS-BSA to the tubes. Centrifuge the tubes for 5 min at 4 &#176;C at 430 x g. Remove and discard the supernatant. Repeat.</li>
	<li>In the meantime, prepare a 1:10 dilution of streptavidin-phycoerythrin diluted in PBS-BSA buffer. Pipette 20 &#181;L of the 1:10 dilution of streptavidin-phycoerythrin and add to the tubes. Mix the tubes gently and incubate for 45 min at 4 &#176;C.</li>
	<li>Add 750 &#181;L of PBS-BSA buffer into the tubes. Mix well and centrifuge for 5 min at 4 &#176;C at 430 x g. Remove and discard the supernatant. Repeat.</li>
	<li>During centrifugation, prepare a 1:100 dilution of biotinylated anti-streptavidin antibody diluted in PBS-BSA buffer.</li>
	<li>Once the centrifugation is done, remove and discard the supernatant.</li>
	<li>Pipette 20 &#181;L of the 1:100 dilution of biotinylated anti-streptavidin into the tubes. Mix the tubes gently. Incubate the tubes for 45 min at 4 &#176;C.</li>
	<li>After 45 min of incubation, pipette 750 &#181;L of PBS-BSA buffer and add into the tubes. Centrifuge the tubes for 5 min at 4 &#176;C at 430 x g. Remove and discard the supernatant. Repeat.</li>
	<li>Pipette 20 &#181;L of the 1:10 dilution of streptavidin-phycoerythrin and add into the tubes. Mix the tubes gently. Incubate the tubes for 45 min at 4 &#176;C.</li>
	<li>Pipette 750 &#181;L of PBS-BSA buffer and add into the tubes. Mix well and centrifuge the tubes for 5 min at 4 &#176;C at 430 x g. Remove and discard the supernatant. Repeat this step 2x.</li>
</ol>
4. Immunostained erythrocyte fixation
<ol>
	<li>During the last centrifugation, prepare the fixation buffer, a 1:100 dilution of 37% formaldehyde using the washing buffer PBS-BSA.</li>
	<li>Pipette 450 &#956;L of fixation buffer and add into immunostained erythrocyte tubes (from step 3.13) while vortexing for 5 s.</li>
	<li>Pipette all fixed cells into 5 mL round bottom tubes and store in the refrigerator. 
	NOTE: The protocol can be paused here for up to 48 h.</li>
</ol>
5. Flow cytometry analysis of stained erythrocytes
NOTE: It is advisable to refer to the operator&#39;s manual for the cytometer (see Table of Materials) to know how to perform the cytometric readings. The suggested parameters below apply to the instrument used and must be optimized for each cytometer.
<ol>
	<li>Turn on the flow cytometer, then turn on the computer. Let the optical system temperature stabilize by leaving it on for 30 min. Check the cytometer window in the software to ensure that the cytometer is connected to the workstation (the message Cytometer Connected is displayed).</li>
	<li>Check that the buffer container is full and that the waste container is empty. Remove air bubbles in the buffer filter and the buffer line using the purge system. Prime the fluidics system by pressing the Prime button on the console of the cytometer. Wait until the indicator light changes from red to green.</li>
	<li>To clean the fluidics, install a tube containing 3 mL of a cleaning solution on the sample injection port and allow the cleaning solution to run for 5 min with a high sample flow rate. Repeat this with the rinse solution with distilled water. Leave the tube containing water on the sample injection port.</li>
	<li>To prepare the calibrating beads, pipette 400 &#181;L of PBS into the bottom of a round bottom tube. Mix the bead stocks strongly by vortexing for 30 s. Add a drop to the round bottom tube containing PBS. Mix carefully by vortexing for 30 s.</li>
	<li>Run the performance check. Open the cytometer Setup and Tracking module in the software (Figure 1A). Verify that the cytometer configuration is correct for the experiment using PE immunostaining. Verify that the calibrating bead batch is correct with the configuration.</li>
	<li>Install the bead tube on the sample injection port and let it run with a low sample flow rate. Run the performance check, which takes approximately 5 min to complete). Once the performance check is complete, verify that the cytometer performance is satisfactory (Figure 1B). Close the cytometer Setup and Tracking module in the software.</li>
	<li>To set up an experiment and create application settings, click the New Experiment button on the browser toolbar and open the new experiment. Specify the parameters by selecting appropriate cytometer settings: Forward Scatter (FSC), Side Scatter (SSC), and PE from the drop-down menu of the experiment (Figure 1C). Select Linear Mode for FSC parameter and Logarithm Mode for SSC and PE parameters.</li>
	<li>In the open experiment, select Cytometer Settings (Figure 1D), then select Application Settings, and create a global worksheet (Figure 1E). Use the gray boxes and crosshairs to guide the optimization.</li>
	<li>Load the unstained control tube onto the cytometer and run Acquisition. Ensure that the population of interest (i.e., RBCs) is on scale by optimizing the FSC and SSC voltages. Optimize the FSC threshold value to eliminate debris without interfering with the population of interest.</li>
	<li>Draw a gate around the RBCs on the FSC vs. SSC plot. Display the RBC population in the dot plot of PE fluorescence. If needed, increase the fluorescence of the photomultiplier tube (PMT) voltages to place the negative population within the gray boxes. Unload the unstained control tube from the cytometer.</li>
	<li>Verify that the positive populations are on scale. Load the stained control tube onto the cytometer and run Acquisition. Lower the PMT voltage for the positive population if it is off scale until the positive population can be seen entirely on scale. Then unload the stained sample.</li>
	<li>To record and analyze samples, on a new global worksheet, create the following plots for previewing the data: 1) FSC vs. SSC, and 2) PE fluorescence histogram. Load the first sample onto the cytometer and run Acquisition.</li>
	<li>Draw an RBC gate around the erythrocytes on the FSC vs. SSC plot. Display the RBC population in the PE fluorescence histogram. In the Statistics view, select the mean for PE fluorescence parameters on GR populations (Figure 1F).</li>
	<li>In the Acquisition dashboard, select all events in the stopping gate and 10,000 events to record (Figure 1G). Click Record Data. When the event recording has completed, remove the first tube from the cytometer. The global worksheet plots should look like those in Figure 2.</li>
	<li>Load the following samples and record them.</li>
</ol>
6. Determination of the density of erythrocyte CR1
<ol>
	<li>Take the values of the mean fluorescence intensities of the samples corresponding to the &#34;low&#34; subject (Figure 3, Table I, RBC MFI), &#34;medium&#34; subject (Figure 4, Table D, RBC MFI), &#34;high&#34; subject (Figure 4, Table I, RBC MFI), and to the negative control sample (Figure 3, Table I, RBC MFI).</li>
	<li>On a graph representing the mean fluorescence intensity as a function of the density of CR1, place the four points corresponding to the negative control, &#34;low&#34; subject, &#34;medium&#34; subject, and &#34;high&#34; subject (blue points, Figure 6).</li>
	<li>Draw the regression line to get the calibration line and its equation.</li>
	<li>Take the values of the mean fluorescence intensity of the samples corresponding to the subjects whose density is to be determined. (Figure 5, Tables D and I, RBC MFI).</li>
	<li>Obtain the equation by replacing &#34;Y&#34; using the values of the mean fluorescence intensities, and calculate the density of CR1/E (Figure 6).</li>
	<li>Check on the graph that the mean fluorescence intensity values and the determined CR1/E density correspond to a point on the calibration line (Figure 6).</li>
</ol>

The authors have nothing to disclose.

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).

<table border="0" cellpadding="0" cellspacing="0" style="width:752px;" width="753">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<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>
		</tr>
		<tr height="63">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Blouse</td>
			<td style="width:239px;"></td>
			<td style="width:119px;"></td>
			<td>protection</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Comorack-96</td>
			<td style="width:239px;">Dominique DUTSCHER SAS, F-67172 Brumath</td>
			<td style="width:119px;">944060P</td>
			<td>rack</td>
		</tr>
		<tr height="48">
			<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>
		</tr>
		<tr height="48">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="48">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="48">
			<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>
		</tr>
		<tr height="48">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="48">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
	</tbody>
</table>

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, ...

measuring-erythrocyte-complement-receptor-1-using-flow-cytometry

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JoVE Journal

Immunology and Infection

7.0K Views.  Reims University Hospitals, Maison Blanche Hospital. 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).

Video: Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH50 is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zero; if one or more components of the classical pathway are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...