This research was funded by the Canadian Blood Services (CBS) and the Canadian Institutes of Health Science (CIHR) Research Partnership Fund. The authors thank the LMB Macromolecule Hub at the UBC Centre for Blood research for the use of their research facilities. The infrastructure facility is supported by the Canada foundation for Innovation (CFI) and the Michael Smith Foundation for Health Research (MSFHR). R. Chapanian is a recipient of (CIHR/CBS) postdoctoral fellowships in Transfusion Science and a recipient of MSFHR research trainee post doctoral fellowship. J.N. Kizhakkedathu is a recipient of MSFHR Career Investigator Scholar Award.

A. Hyperbranched Polyglycerol Modification (SS-HPG)
<ol>
 <li>Place lyophilized HPG 60 kDa (0.5 g, 0.0083 mmol) in a round bottom flask and dry it overnight under vacuum at 90 &deg;C.</li>
 <li>Refrigerate the flask to room temperature, and dissolve the dried HPG in anhydrous pyridine (3 ml).</li>
 <li>To functionalize approximately eight hydroxyl groups on HPG with carboxyl groups, add catalytic amount of dimethylaminopyridine (one drop of 5 mg/ml solution in pyridine) to the HPG solution. To this mixture, add succinic anhydride, (0.0067 g, 0.0664 mmol) dissolved in 0.5 ml pyridine drop wise over 10 min. Stir the mixture overnight at room temperature under argon.</li>
 <li>Precipitate the mixture in 40 ml of cold acetone (4 &deg;C) in a 50 ml centrifuge tube, and centrifuge using a Beckman J2-MC centrifuge at 27,000 x g for 15 min. Decant the supernatant, and remove residual acetone by flushing with argon at room temperature.</li>
 <li>To activate the carboxyl groups with succinimidyl succinate (SS) groups, dissolve the carboxyl -functionalized HPG in 3 ml of anhydrous DMF. Add N-hydroxysuccinimide (0.0077 g, 0.0664 mmol) and N, N'-diisopropylcarbodiimide (0.0084 g, 0.0664 mmol) to the HPG solution and stir the mixture overnight at room temperature under argon.</li>
 <li>Purify the SS-HPG by precipitation in cold acetone.</li>
 <li>Remove residual acetone by flushing with argon.</li>
 <li>Determine the purity of modified HPG and the degree of carboxyl and SS functionalization by proton (1H)- NMR analysis.</li>
</ol>
B. Whole Blood Collection and Separation of Red Blood Cells (RBCs)
<ol>
 <li>Collect whole blood (40% Hematocrit, 3 ml) from consented healthy human donors into a citrate vacutainer tube.</li>
 <li>Centrifuge the citrate tube at 1,000 x g for 4 min.</li>
 <li>Remove the supernatant that contains plasma and platelets, and the intermediate buffy coat that contains white blood cells and platelets using a Pasteur pipette.</li>
 <li>Transfer packed RBCs (80% Hematocrit, 1.2 ml) into a 12 ml Falcon centrifuge tube, and add PBS buffer (8 ml, pH = 8.0) to wash RBCs.</li>
 <li>Mix RBCs by inversion to obtain uniform cell distribution.</li>
 <li>Centrifuge the Falcon tube at 1,000 x g for 4 min, and remove the supernatant.</li>
 <li>Wash RBCs with PBS two more times by repeating steps 5 and 6.</li>
 <li>Place packed-RBCs (100 &mu;l) in 1.6 ml Eppendorf tube, and add PBS buffer (300 &mu;l, pH = 8.0) to obtain 20 % Hematocrit RBCs.</li>
</ol>
C. HPG Grafting to RBCs
<ol>
 <li>Immediately after precipitation of modified HPG in acetone in step A6, place SS-HPG (150 mg) in a glass vial (1 dram), and dissolve it in PBS buffer (300 &mu;l, pH = 8.0).</li>
 <li>Add SS-HPG polymer solution into 20% Hematocrit washed RBCs to obtain a final volume of 400 &mu;l and SS-HPG concentration that ranges from 0.5 mM to 3 mM. For example to prepare RBCs treated with 0.5 mM SS-HPG, remove 36 &mu;l of PBS from the washed RBC and place 36 &mu;l of SS-HPG polymer solution.</li>
 <li>Vortex the suspension gently and place the Eppendorf tubes on an orbital shaker at room temperature for 1 hr.</li>
 <li>Centrifuge the Eppendorf tubes at 1,000 x g for 4 min, and pipette out the supernatant.</li>
 <li>Add 1 ml of PBS to wash RBCs, centrifuge and remove the supernatant.</li>
 <li>Add 1 ml saline and wash RBCs twice as in step 5.</li>
 <li>Add 300 &mu;l of saline to 100 &mu;l of packed RBCs (80% Hematocrit) to a final concentration of 20 % Hematocrit.</li>
</ol>
D. Characterization of HPG Modified RBCs
I. Complement mediated lysis
<ol>
 <li>Collect around 8 ml of whole blood from healthy human donors into BD vacutainer glass serum tube, and allow the blood to clot at room temperature for 30 min.</li>
 <li>Centrifuge the tube at 2,000 x g for 10 min and collect around 3 ml of serum.</li>
 <li>One milliliter of the serum was placed in a water bath at 60 &deg;C for 30 min to prepare heat inactivated serum.</li>
 <li>Add 60 &mu;l of fresh serum or heat inactivated serum into an Eppendorf tube that contain 60 &mu;l of 20% Hematocrit HPG modified RBCs or unmodified RBCs.</li>
 <li>Incubate RBCs for 1 hr at 37 &deg;C.</li>
 <li>To quantify the amount of hemoglobin in the cell suspension, place 5.9 &mu;l of 20% hematocrit of HPG modified RBCs or unmodified cells in triplicate in 96 well plate. Add 294 &mu;l of Drabkin's reagent (a reagent that is used to quantify the amount of hemoglobin spectrophotomerically). Mix cells with Drabkin's reagent by pipetting in and out. Measure the absorbance of the hemoglobin cyanoderivative at 540 nm using SPECTRA MAX 190 plate reader.</li>
 <li>To quantify the amount of hemoglobin in the supernatant, centrifuge cells at 13,000 x g for 1 min. Place 50 &mu;l of supernatant in triplicate in a 96 well plate. Add 250 &mu;l of Drabkin's reagent. Mix cells with Drabkin's reagent by pipetting in and out. Measure the absorbance of the hemoglobin cyanoderivative at 540 nm using SPECTRA MAX 190 plate reader.</li>
 <li>Quantify the amount of lysed cells from the ratio of hemoglobin in the supernatant (step 7) and the total hemoglobin in the sample (step 6) 8, 11, 12.</li>
</ol>
II. The camouflage of major and minor antigens using MTS cards
<ol>
 <li>Place 50 &mu;l of HPG modified RBC (10 % Hematocrit) in an Eppendorf tube, centrifuge at 1,000 x g for 1 min, and remove the supernatant.</li>
 <li>Add 110 &mu;l of MTS diluent to the cell pellet and homogenize the cell suspension by pipetting in and out.</li>
 <li>Add 11 &mu;l of cell suspension to each mini gel column. Avoid touching the gel during the addition.</li>
 <li>Locate MTS cards into a centrifuge card holder, and centrifuge at 156 x g for 6 min.</li>
 <li>Protection of surface antigens is determined from the location of RBC in the mini gel column, based on the manufacturer description.</li>
</ol>
III. Aqueous two phase partition measurements
<ol>
 <li>Prepare Dextran 500 kDa/PEG 8 kDa two phase separation system composed of (5% dextran 500k, 4% PEG 8K, 150 mM NaCl, 10 mM phosphate) according to 13.</li>
 <li>Centrifuge at 433 x g for 10 min at room temperature to obtain a two phase system (dextran in the bottom).</li>
 <li>Separate the two phases carefully.</li>
 <li>Aliquot 1.0 ml of the upper PEG phase into a labelled tube (with concave bottom), add 20 &mu;l of 20% hematocrit HPG modified RBC, and mix cells gently by flicking.</li>
 <li>Add 0.5 ml of the upper phase that contain cells to 0.5 ml of a lower phase, and mix the system gently by flicking.</li>
 <li>Place the tube on the bench for ~ 2 min for the system to separate, and determine the location of cells in the system. Localization of RBCs (in the upper PEG phase, in the lower dextran phase or in both) is function to the degree of modification and molecular weight of HPG.</li>
</ol>
IV. Osmotic fragility measurements
<ol>
 <li>Prepare NaCl solutions with concentrations that range from 0 to 0.9 (w/v) %.</li>
 <li>Place 0.4 ml of NaCl solutions into 1.5 ml Eppendorf tubes.</li>
 <li>Add 20 &mu;l of RBCs (20% hematocrit) into the NaCl solutions.</li>
 <li>Mix cells carefully by inversion and place them in a water bath at 37 &deg;C for 30 min.</li>
 <li>Suspend cells carefully by inversion. Take 50 &mu;l of RBC suspension and add it to a 1 ml Drabkin's reagent, placed in a cuvette. Mix cells with Drabkin's reagent by pipetting in and out. Measure the absorbance of the hemoglobin cyanoderivative at 540 nm using BECKMAN COULTER DU 730 UV/VIS reader.</li>
 <li>To quantify the amount of hemoglobin in the supernatant, centrifuge cells at 1,000 x g for 1 min. Add 200 &mu;l of supernatant to 1 ml of Drabkin's reagent in a cuvette. Mix cells with Drabkin's reagent by pipetting in and out. Measure the absorbance of the hemoglobin cyanoderivative at 540 nm using BECKMAN COULTER DU 730 UV/VIS reader.</li>
 <li>Calculate the amount of cells lysed in different NaCl solution from the ratio of the amount hemoglobin in the supernatant (step 6) to the total amount of hemoglobin in the cell suspension (step 5).</li>
</ol>
V. Flow cytometry measurements - Protection of Rhesus-D (RhD) antigen
<ol>
 <li>Add 5 &mu;l of control or HPG modified RBCs (20% hematocrit) in triplicate to PBS buffer supplemented with 0.5% BSA to a total volume of 25 &mu;l.</li>
 <li>Add 25 &mu;l of FITC monoclonal anti-Rhesus D (RhD) purchased from Quotient Biodiagnostics (PA, USA).</li>
 <li>Incubate the mixture for 30 min at 37 &deg;C in a water bath.</li>
 <li>Wash the mixture with 1.5 ml PBS buffer two times. Centrifuge at 1,000 x g for 1 min to remove the supernatant.</li>
 <li>Suspend RBCs into 1 ml saline, and transfer the suspension into 4 ml flow BD flow cytometry tube.</li>
 <li>Analyze using FACSCanto II flow cytometer, acquiring 5,000 events on the RBC control gate, and use whole events for analysis.</li>
</ol>
VI. Flow cytometry measurements - Expression of CD47
<ol>
 <li>Add 1.54 &mu;l of control or HPG modified RBCs (20 % hematocrit) in triplicate to PBS buffer supplemented with 0.5% BSA to a total volume of 44 &mu;l.</li>
 <li>Add 6 &mu;l of phycoerythrin (PE) labelled mouse anti-human CD47 purchased from BD Biosciences (NJ, USA).</li>
 <li>Incubate the mixture for 30 min at 37 &deg;C in a water bath.</li>
 <li>Wash the mixture with 1.5 ml PBS buffer for two times. Centrifuge at 1,000 x g for 1 min to remove the supernatant.</li>
 <li>Suspend RBCs into 1 ml saline, and pass through 26 G 5/8 needle to minimize cell clumping.</li>
 <li>Transfer the suspension into 4 ml flow BD flow cytometry tube.</li>
 <li>Analyze using FACSCanto II flow cytometer, acquiring 10,000 events on the RBC control gate.</li>
</ol>

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No conflicts of interest declared.

Universal donor RBCs have great potential in enhancing blood availability and safety for blood transfusion therapy. RBCs are also considered promising drug delivery vehicles due to their long circulation and inherent biocompatibility 14, 15. Experiments presented in this paper evaluate the in vitro characteristics of HPG modified RBCs. The in vitro properties and in vivo circulation of HPG modified RBCs have been investigated in our group recently 8, 11. The partition...

<table border="1">
 <tbody>
 <tr>
 <td>Glycidol </td>
 <td>Sigma Aldrich </td>
 <td></td>
 <td>(ON, Canada) </td>
 </tr>
 <tr>
 <td>Trimethylolpropane </td>
 <td>Fluka </td>
 <td></td>
 <td>(ON, Canada) </td>
 </tr>
 <tr>
 <td>Potassium methylate</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>Anhydrous pyridine</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>4-Dimethylaminopyridine</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>Succinic anhydride</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>Anhydrous dimethyl formamide</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>N-Hydroxysuccinimide </td>
 <td>Sigma Aldrich </td>
 <td></td>
 <td>(ON, Canada) </td>
 </tr>
 <tr>
 <td>N,N'-Diisopropylcarbodiimide </td>
 <td>Sigma Aldrich </td>
 <td></td>
 <td>(ON, Canada) </td>
 </tr>
 <tr>
 <td>MTS cards</td>
 <td></td>
 <td></td>
 <td>Micro Typing System (MTS) cards (FL, USA)</td>
 </tr>
 <tr>
 <td>Dextran 500 kDa</td>
 <td>Pharmacia Fine Chemicals</td>
 <td></td>
 <td>(Sweden)</td>
 </tr>
 <tr>
 <td>PEG 8 kDa</td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td>FITC monoclonal anti-Rhesus D (RhD)</td>
 <td>Quotient Biodiagnostics</td>
 <td></td>
 <td>(PA, USA)</td>
 </tr>
 <tr>
 <td>PE monoclonal anti-CD47 </td>
 <td>BD Biosciences </td>
 <td></td>
 <td>(NJ, USA) </td>
 </tr>
 <tr>
 <td>Drabkin's reagent </td>
 <td>Sigma Aldrich</td>
 <td></td>
 <td>(ON, Canada)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table. Chemicals and reagents used for the grafting of HPG polymers to RBC membrane and their analysis.</td>
 </tr>
 </tbody>
 </table>

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.

Camouflage of Rhesus D antigen and CD47 RBC surface protein were quantified by flow cytometry using fluorescent labelled monoclonal antibodies, and a representative result is given in Figure 1. In case of HPG-grafted RBCs (grey), the intensity of the signal decreased (peak shifted to left) compared to the control RBCs (red &amp; green) indicating a reduction in binding to antibodies to cell surface which indicate the masking of surface proteins.
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Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

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 ...

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Immunology and Infection

18.0K Views.  University of British Columbia. 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.

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

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 ...

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

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 ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.