The author would like to thank Miss Lora Matthews for performing the technique and Mr Paul Shepherd for cinematography. This project was funded by the University of South Australia, Laboratory Medicine program.

Preparation of 5x Veronal Buffered Saline (VBS)
<ol>
	<li>To prepare the Veronal Buffered Saline (VBS), three separate solutions need to be prepared.</li>
	<li>Prepare solution 1 by dissolving 21.25gm of NaCl and 0.94gm of Sodium Barbitone in 350ml of distilled water. The final concentrations of NaCl and Sodium Barbitone are 1.02M and 13mM respectively.</li>
	<li>Prepare solution 2 by dissolving 1.44gm of Barbitone in 125ml of hot distilled water. The final concentration of Barbitone is 62.5mM.</li>
	<li>Prepare solution 3 by dissolving 20.33gm of MgCl2 and 4.41gm of CaCl2 in 100ml of distilled water. The final concentration of MgCl2 and CaCl2 is 2.18M and 440mM respectively.</li>
	<li>Mix solutions 1 and 2 and cool to room temperature.</li>
	<li>Once the combined solution has cooled, add 1.25ml of solution 3 and adjust the pH to 7.3-7.5 using 1M HCl.</li>
	<li>Adjust the final volume to 500ml with distilled water to prepare a 5x stock solution.</li>
	<li>To prepare a 1x working solution, dilute the stock 1:5 with distilled water.</li>
</ol>
Sensitisation of sheep red blood cells with haemolysin
<ol>
	<li>Prepare the haemolysin by firstly diluting it 1:50 with VBS</li>
	<li>To 4ml of SRBC add 6ml of VBS and gently mix by inversion</li>
	<li>Centrifuge at 600g x 5minutes</li>
	<li>Discard the supernatant and wash the cells another 2 times</li>
	<li>After the final wash, centrifuge the cells at 900g x 5minutes to pack the cells</li>
	<li>Discard the supernatant and resuspend the cells in sufficient VBS to prepare a 10% solution i.e. 0.5ml of packed cells are resuspended in 5 ml of buffer</li>
	<li>Dropwise add an equal volume of haemolysin (rabbit anti-sheep red blood cell antibody) to 	the cells while swirling continuously</li>
	<li>Incubate at 30&deg;C for 30 minutes in a water bath</li>
	<li>Gently mix the cells every 15minutes</li>
	<li>Sensitised SRBC can be stored overnight at 4&deg;C</li>
</ol>
CH50 assay
<ol>
	<li>Label a series of tubes in duplicate with 1:8, 1:16, 1:32, 1:64 and 1:128.</li>
	<li>Prepare a series of two fold serial dilutions of control and test serum in VBS each in duplicate</li>
	<li>Start at 1:4 (100ml serum + 300ml VBS) and transfer 200ml of sample into the next labelled tube.</li>
	<li>Mix thoroughly between dilutions and transfer 200ml to the next dilution with a fresh pipette tip.</li>
	<li>Repeat until all five dilutions are made.</li>
	<li>Discard 200ml from the final 1:128 dilution.</li>
	<li>Add 200ml of suspended sensitised SRBC to all tubes.</li>
 <li>Label two separate tubes as BLANK and add 200ml of sensitised SRBC + 200ml VBS. These tubes will measure spontaneous lysis of the SRBC in the VBS.</li>
 <li>Label another two separate tubes as TOTAL LYSIS and add 200ml of sensitised SRBC + 200ml distilled water.</li>
	<li>Gently mix all tubes.</li>
	<li>Incubate at 37&deg;C for 30 minutes in a waterbath mixing after 15 minutes.</li>
	<li>Centrifuge the samples at 1,500g for 5 minutes to sediment the RBCs.</li>
	<li>Transfer 100ml of supernatant from each tube to a well in a 96 well flat bottom plate.</li>
	<li>Add 100ml of distilled water to each well.</li>
	<li>Read the absorbance of the samples at 540nm using a plate spectrophotometer.</li>
</ol>
Calculations
<ol>
	<li>Calculate the mean absorbance for each sample</li>
	<li>Subtract the BLANK absorbance (spontaneous lysis) from all samples</li>
	<li>Calculate the % lysis for each dilution using the following formula: <img src="/files/ftp_upload/1923/1923eq1.jpg" alt="Equation" /></li>
	<li>Plot the percentage lysis (vertical axis) versus the serum dilution on the horizontal axis.</li>
	<li>Calculate the dilution required for 50% haemolysis for the control and test serum (Fig 2).</li>
</ol>
Representative Results:
The video includes an example of representative results. In practice, a control serum is also run at the same time as the test serum and is treated in the same manner. Below (Table 1) is sample data from a tested serum sample. The data is manipulated according to the equation presented in 5.3.
<table border="1">
 <tbody>
 <tr>
 <td>Sample</td>
 <td>OD540</td>
 <td>Mean</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Blank</td>
 <td>0.042, 0.044</td>
 <td>0.043</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Total lysis</td>
 <td>0.183, 0.183</td>
 <td>0.183</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td> 
 </tr>
 <tr>
 	<td colspan="5">&nbsp;</td>
 </tr>
 <tr>
 <td>Dilution</td>
 <td>OD540</td>
 <td>Mean</td>
 <td>Mean-Blank</td>
 <td>% Lysis</td>
 </tr>
 <tr>
 <td>1:8</td>
 <td>0.200, 0.219</td>
 <td>0.210</td>
 <td>0.168</td>
 <td>116</td>
 </tr>
 <tr>
 <td>1:16</td>
 <td>0.179, 0.173</td>
 <td>0.176</td>
 <td>0.134</td>
 <td>93</td>
 </tr>
 <tr>
 <td>1:32</td>
 <td>0.134, 0.110</td>
 <td>0.122</td>
 <td>0.08</td>
 <td>55</td>
 </tr>
 <tr>
 <td>1:64</td>
 <td>0.053, 0.066</td>
 <td>0.059</td>
 <td>0.017</td>
 <td>12</td>
 </tr>
 <tr>
 <td>1:128</td>
 <td>0.044, 0.045</td>
 <td>0.045</td>
 <td>0.003</td>
 <td>2</td>
 </tr>
 </tbody>
</table>
<img src="/files/ftp_upload/1923/1923fig1.jpg" alt="Figure 1" /> Figure 1. Activation of the classical complement pathway. The classical pathway is activated by binding immunoglobulin-M (IgM) or immunoglobulin-g (IgG) on the surface of a target cell. The Fc portion of the Ab binds to C1q, C1r is activated and this in turn activates another molecule of C1r which together activate two molecules of C1s. C1s now cleaves C4 which exposes the binding site for C2 which is also cleaved. The binding of C4b and C2a leads to the formation of a complex referred to as a C3 convertase. This complex now cleaves C3 forming C3a and C3b, some of which combine with the C3 convertase forming a C5 convertase. This complex now acts on C5, with the resulting C5b binding to C6 initiating the formation of the membrane attack complex (MAC). C5b6 acts on C7, which in turn act on C8 and ultimately on C9 resulting in the formation of the final MAC. (Goldsby et al, 2003).
<img src="/files/ftp_upload/1923/1923fig2.jpg" alt="Figure 2" /> Figure 2. Plotting of sample data and calculating the CH50 for a control and test serum sample. (A), The calculated percentage (%) lysis of both the control (&bull;) and test () serum samples are plotted against dilution factor. (B) To calculate the 50% lysis, a line is drawn from the 50% percent value until it intersects with the graph lines and then a vertical line is drawn down to the dilution. In this example, a 35-fold dilution of the control and 21.6-fold dilution of the test serum are required to achieve 50% lysis. This data indicates that there is less complement present in the test serum compared to the control as it required less dilution before 50% lysis was reached. The control sample also shows &gt; 100% lysis at the 1:8 dilution. This is most likely due to incomplete lysis of the SRBC by the Distilled water and more efficient lysis by the complement components.

<ol>
	<li>Goldsby, R. A., Kindt, T. J., Osborne, B. A., Kuby, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunology. , (2003).</li></ol>

The CH50 assay is subject to many interferences. Some SRBC are more fragile than others, resulting in spontaneous haemolysis that is unrelated to complement activity. The affinity of the rabbit antibody varies from lot to lot and from one manufacturer to another; this affects the amount of antibody that binds to the SRBC. Also, the process of sensitising SRBC with antibody results in cells with differing amounts of antibody coating the SRBC. Specimen collection and storage are an important potential source ...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sheep red blood cells</td>
<td>&nbsp;</td>
<td>CSL</td>
<td>2490201</td>
<td>Use at 1% final</td>
</tr>
<tr>
<td>Serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Human serum</td>
</tr>
<tr>
<td>Rabbit Anti-Sheep Haemolytic serum (RBCs), Unconjugated</td>
<td>&nbsp;</td>
<td>AdB Serotec</td>
<td>C12HSB</td>
<td>&nbsp;</td>
<tr>
<td>96 well flat bottom plate</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>83.1839</td>
<td>&nbsp;</td>
<tr>
<td>Veronal buffered saline</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use at 1x final</td>
</tr>
<tr>
<td>Waterbath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plate reader</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>37&deg;C room/incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Measuring the 50% Haemolytic Complement CH50 Activity of Serum at JoVE.com

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.

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 ...

measuring-the-50-haemolytic-complement-ch50-activity-of-serum

Research

JoVE Journal

Immunology and Infection

37.5K Views.  University of South Australia. 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.

Video: Measuring the 50% Haemolytic Complement CH50 Activity of Serum

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

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, ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

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 ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...