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

7.1K Views.  Reims University Hospitals, Maison Blanche Hospital. Dit protocol kan worden gebruikt om het aantal antigene sites op cellulaire receptor van belang te meten. Het belangrijkste voordeel van deze techniek is dat het robuuste resultaten produceert, zelfs voor receptoren uitgedrukt bij een lage dichtheid. Deze methode maakt de evaluatie van de vermindering van CR1 erythrocyte expressie en dit is zoals de ziekte van Alzheimer, systemische lupus erythematosus, AIDS, en malaria.Dit protocol is nuttig voor elke analyse van de dichtheid van de c...

Video: Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

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, ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...

Developmental Biology

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Biology

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

Measuring Microglial Uptake of Intravitreally-Delivered Fluorescent Particles Using Flow Cytometry

Source: Murinello, S. et al., <a href="https://app.jove.com/v/54677">Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay.</a> J. Vis. Exp.&#160;(2016)
This video demonstrates flow cytometry for measuring microglial uptake of fluorescent bioparticles after intravitreal delivery in a mouse eye. Flow cytometry detects live cells expressing CD11b and distinguishes phagocytic microglia cells exhibiting green fluorescence from other myeloid cells, including Ly6C-expressing monocytes and Ly6G-expressing neutrophils.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

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