The authors acknowledge funding through the Department of Defense Congressionally Directed Medical Research Programs (#W81XWH-10-1-0445), National Institutes of Health (NIH R21 HL110056), and American Heart Association (#11SDG4890030).

1. Preparation and Sterilization of Buffers and Test Agents
<ol>
 <li>150 mM NaCl buffer: Dissolve 4.383 g NaCl crystals in 500 ml of nanopure water.</li>
 <li>pH Buffers: Prepare phosphate buffers at pH 5.6, 6.2, 6.8, and 7.4 by mixing appropriate amounts of monobasic and dibasic sodium phosphate. If samples are to be tested at lower pH values (i.e. pH &lt; 5.6) then a more appropriate buffer, such as citrate buffer, should be used. Buffer recipes are readily available, and an example reference has been provided here.12 </li>
 <li>Sterilize all buffers noted above through a bottle-top vacuum filtration apparatus and re-check buffer pH's. </li>
 <li>20% Triton X-100 (positive control): Mix 20 ml pure Triton X-100 in 80 ml of nanopure water. Vortex vigorously and sonicate to dissolve. Leave at room temperature overnight before use. </li>
</ol>
2. Preparation of Erythrocytes
<ol>
 <li>Obtain 25 ml of blood from an anonymous human donor, drawn directly into K2-EDTA-coated Vacutainer tubes to prevent coagulation. </li>
</ol>
NOTE: All procedures must be pre-approved by the appropriate Institutional Review Board (IRB), and venipuncture and blood collection must be performed by a trained phlebotomist in order to minimize the risk to the donor. Standard phlebotomy procedures have been published elsewhere.13
<ol start="2">
 <li>Centrifuge blood at 500 x g for 5 min, and mark levels of hematocrit (red, lower layer) and plasma (yellowish, upper layer) on tube.</li>
 <li>Aspirate plasma gently via a micropipettor, add into bleach, and discard into biohazardous waste. </li>
 <li>Fill hematocrit tube to marked line (original level of plasma) with 150 mM NaCl solution. Cap and invert a few times to gently mix. Centrifuge at 500 x g for 5 min. </li>
 <li>Repeat step 2.3-2.4 to wash blood cells again. Then aspirate supernatant and replace with PBS at pH 7.4. Invert to mix. </li>
 <li>Split blood evenly into four tubes, corresponding to each pH that will be tested. Label the tubes according to each pH to be tested (5.6, 6.2, 6.8, 7.4).</li>
 <li>Centrifuge blood tubes at 500 x g for 5 min. Mark levels on tubes, then aspirate supernatant.</li>
 <li>Fill each tube to marked line with buffer of appropriate pH (as indicated in 2.6). </li>
 <li>Label four 50 ml conical tubes (one per pH to test), and pipet 49 ml of PBS of appropriate pH into each conical tube. </li>
 <li>Add 1 ml of erythrocytes (same pH) into corresponding tube for a 1:50 dilution. Visually inspect the diluted blood, which should be turbid and will settle if left undisturbed. If no pellet forms, cells have lysed. </li>
</ol>
3. Lysis Assay 96 Well Plate Setup and Quantification
<ol>
 <li>Prepare stock solutions of all experimental drug delivery agents, at 20x the desired final concentration to be tested (Assay will take 10 &mu;l of drug delivery agent + 190 &mu;l diluted red blood cells, leading to a 1/20 dilution of the original drug delivery agent into the final test mixture). Stocks of 20, 100, and 800 &mu;g/ml are suggested, resulting in final test concentrations of 1, 5, and 40 &mu;g/ml, respectively.</li>
 <li>Pipet 10 &mu;l of each stock solution into V-bottom 96-well plates. For optimal results, load each sample in triplicate or quadruplicate.</li>
</ol>
NOTE: For ease, it is recommended that a separate 96-well plate should be prepared for each pH to be tested, with each sample (at each concentration) loaded at n=3-4 per plate. 
<ol start="3">
 <li>For positive control wells, add 10 &mu;l of 20% Triton X-100. </li>
 <li>For negative control wells, add 10 &mu;l of phosphate buffer. Use buffer at the same pH to be tested.</li>
 <li>Pipet 190 &mu;l of diluted erythrocytes (see 2.10) to each well, making sure cell stock solution remains homogenous during transfer. Hint: Use multi-channel pipette to simplify this task. </li>
 <li>Incubate plates at 37 &deg;C for one hour (Optional: Use an orbital shaker or rocker). </li>
 <li>Centrifuge plates for 5 min at 500 x g to pellet intact erythrocytes. Note: when removing the plate from the centrifuge and transporting it to the next step, handle with care and be certain not to disrupt the cell pellet. </li>
 <li>Using a multichannel pipet, transfer 100 &mu;l of supernatant from each well into a clear, flat-bottomed 96-well plate. Note: if the cell pellet is accidentally disturbed for any sample(s), one can re-centrifuge the plate and then proceed with supernatant transfer. </li>
 <li>Measure absorbance of supernatants with a plate reader. Note that a range of wavelengths can be used (400 - 541 nm). </li>
</ol>
NOTE: Different plate readers may have different saturation points and sensitivities, so the choice of a wavelength for measurements depends on whether or not hemolysis data from experimental samples can be reliably normalized against maximum hemolysis as induced by detergent treatment. This requires accurate measurement of absorbance values of the positive control samples. 
<ol start="10">
 <li>Using Microsoft Excel or a similar data analysis software, find the average of the background absorbance readings from the negative control samples set up for each pH (step 3.4). Subtract this background absorbance value from all other samples that were measured at that pH.</li>
 <li>After background subtraction, find the average absorbance of the positive control detergent-treated samples (step 3.3). Then normalize all experimental data points to this mean absorbance value, which should represent 100% hemolysis. Finally, multiply each well value by 100% to calculate % hemolysis that occurred in each individual well relative to the detergent control.</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolytic+Activity+of+pH-Responsive+Polymer-Streptavidin+Bioconjugates.">Hemolytic Activity of pH-Responsive Polymer-Streptavidin Bioconjugates.</a> Bioconjugate Chemistry. 10, 401-405 (1999).</li><li>Murthy, N., Campbell, J., Fausto, N., Hoffman, A. S., Stayton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinspired+pH-responsive+polymers+for+the+intracellular+delivery+of+biomolecular+drugs.">Bioinspired pH-responsive polymers for the intracellular delivery of biomolecular drugs.</a> Bioconjugate chemistry. 14, 412-419 (2003).</li><li>Murthy, N., Robichaud, J. R., Tirrell, D. A., Stayton, P. S., Hoffman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+and+synthesis+of+polymers+for+eukaryotic+membrane+disruption.">The design and synthesis of polymers for eukaryotic membrane disruption.</a> Journal of controlled release : official journal of the Controlled Release Society. 61, 137-143 (1999).</li><li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+endosomal+barrier+by+amphotericin+B-loaded+dual+pH-responsive+PDMA-b-PDPA+micelleplexes+for+siRNA+delivery.">Overcoming endosomal barrier by amphotericin B-loaded dual pH-responsive PDMA-b-PDPA micelleplexes for siRNA delivery.</a> ACS nano. 5, 9246-9255 (2011).</li><li>Nelson, C. 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IRB approval: Procedures involving human subjects have been approved by the Vanderbilt University Institutional Review Board (IRB; Protocol #111251). No conflicts of interest declared.

pH-responsive polymers or other agents designed for endosomolytic function can be rapidly and effectively screened based on lysis of red blood cells at pH values encountered in the endosome (Figure 1; pH 6.8 - early endosome, pH 6.2 - late endosome, pH 5.6 - lysosome).14-17 pH-dependent hemolysis has been used to screen the ability of carriers to mediate endosomal release of biomacromolecular therapeutics (e.g. peptides, siRNA, ODNs, proteins), and results of this assay can be predict...

Although there are many potential high-impact therapeutic targets inside the cell, the intracellular delivery of agents poses a significant challenge. Frequently, drugs, especially biologics, are internalized by cells and trafficked into vesicles that either lead to degradation of their contents through the endo-lysosomal pathway, or are shuttled back out of the cell via exocytosis.1 In the latter process, the internal pH of the vesicles is acidified to approximately 5-6, which is the optimal pH for activity of enzymes that function in this compartment, such as lysozyme.2 
Recently, a number of materials have been specifically engineered to leverage the acidification of endosomes to facilitate cytosolic delivery of their cargo. One example of this approach uses synthetic, polymer micelle nanoparticles whose core is zwitterionic and charge-neutral at physiologic pH (i.e. 7.4). However, at pH 6.0 - 6.5, the polymers become protonated and acquire a net positive charge that destabilizes the micelle core, and the exposed polymer segments interact with and disrupt the endosomal membrane. This activity has been shown to promote the endosomal escape of peptide and nucleic acid-based therapeutics, allowing them to access their cytosolic targets.3,4 Other examples of methods developed to mediate endosomal escape that disrupt the membrane barrier include 'fusogenic' peptides or proteins that can mediate membrane fusion or transient pore formation in the phospholipid bilayer.5 Homopolymers of anionic alkyl acrylic acids such as poly(propylacrylic acid) are another well-studied approach, and in these polymers, the protonation state of pendant carboxylic acid dictates transition into a hydrophobic, membrane-disruptive state in endo-lysosomal pH ranges.6,7
One useful model system for screening endosomolytic behavior is the ex vivo pH-dependent hemolysis assay.8 In this model system, the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. This generalizable model has been used by others to evaluate the endosomolytic behavior of cell-penetrating peptides and other polymeric gene delivery systems.8-11 In this experiment, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular (7.4), early endosomal (6.8), and late endo-lysosomal (&lt; 6.8) environments. The amount of hemoglobin released during the incubation period is quantified as a measure of red blood cell lysis, which is normalized to the amount of hemoglobin released in positive control samples lysed with a detergent. 
From screening a small library of potentially endosomolytic test materials, one can infer that samples that produce no hemolysis at pH 7.4, but significantly elevated hemolysis at pH &lt; 6.5, will be the most effective and cytocompatible candidates for cytosolic drug delivery. Materials that fit these criteria would be expected to remain inert and not indiscriminately destroy lipid bilayer membranes (i.e. that could cause cytotoxicity) until being exposed to a drop in the local pH following internalization into endo-lysosomal compartments. 
In this protocol, erythrocytes are isolated from a human donor and co-incubated at pH 5.6, 6.2, 6.8, or 7.4 with experimental endosomolytic drug delivery agents. Intact erythrocytes are pelleted, and the supernatants (containing hemoglobin released from lysed erythrocytes) are analyzed for the characteristic absorbance of hemoglobin via a plate reader (Figure 1).

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>BD Vacutainer - K2EDTA 			Vacutainer Tubes</td>
 <td>Fisher Scientific</td>
 <td>22-253-145</td>
 <td>For blood collection</td>
 </tr>
 <tr>
 <td>BD Vacutainer Blood Collection 			Needles, 20.5-gauge</td>
 <td>Fisher Scientific</td>
 <td>02-665-31</td>
 <td>For blood collection</td>
 </tr>
 <tr>
 <td>BD Vacutainer Tube Holder / Needle 			Adapter</td>
 <td>Fisher Scientific</td>
 <td>22-289-953</td>
 <td>For blood collection</td>
 </tr>
 <tr>
 <td>BD Brand Isopropyl Alcohol Swabs</td>
 <td>Fisher Scientific</td>
 <td>13-680-63</td>
 <td>For blood collection</td>
 </tr>
 <tr>
 <td>BD Vacutainer Latex-Free Tourniquet</td>
 <td>Fisher Scientific</td>
 <td>02-657-6</td>
 <td>For blood collection</td>
 </tr>
 <tr>
 <td>Hydrochloric acid (conc.)</td>
 <td>Fisher Scientific</td>
 <td>A144-500</td>
 <td>For adjustment of pH of D-PBS.</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Sigma-Aldrich</td>
 <td>T8787</td>
 <td>Positive control</td>
 </tr>
 <tr>
 <td>Dulbecco's PBS</td>
 <td>Invitrogen</td>
 <td>14190</td>
 <td></td>
 </tr>
 <tr>
 <td>Nalgene MF75 Sterile Disposable 			Bottle-Top Filter Unit with SFCA Membrane</td>
 <td>Fisher Scientific</td>
 <td>09-740-44A</td>
 <td></td>
 </tr>
 <tr>
 <td>BD 96-well plates, flat-bottomed, 			tissue culture-treated polystyrene</td>
 <td>Fisher Scientific</td>
 <td>08-772-2C</td>
 <td>For plate-reading at the end of the 			assay.</td>
 </tr>
 <tr>
 <td>BD 96-well plates, round-bottomed, 			tissue culture-treated polystyrene</td>
 <td>Fisher Scientific</td>
 <td>08-772-17</td>
 <td>For incubation of red blood cells 			with experimental agents.</td>
 </tr>
</table>

ex vivo red blood cell hemolysis assay for the evaluation of ph-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. 
In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Typically, the agents that exhibit ideal pH-dependent hemolytic behavior have the highest potential for cytosolic delivery of drugs, nucleic acids, or other bioactive molecules. This is exemplified by Agent #1 as portrayed in Figure 2, which exhibits minimal hemolysis at pH 7.4, but a sharp increase in hemolytic behavior at endosomal pH ranges (&lt; 6.5). Some agents may exhibit significant levels of hemolytic behavior at physiological pH ranges (Agent #2 at 40 &mu;g/ml; Figure 2), sugge...

Watch this Scientific Journal Video about Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs at JoVE.com

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

ex-vivo-red-blood-cell-hemolysis-assay-for-evaluation-ph-responsive

Research

JoVE Journal

Immunology and Infection

34.0K Views.  Vanderbilt University. A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.

Video: Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

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 ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...