The project was supported by NIH NIAID Contract No. HHSN2662000500021C. We thank Ming Chen for his technical assistance.

1. Program the Amaxa 96 well shuttle Nucleofector 
<ol>
 <li>Open a new parameter file.</li>
 <li>Select the number of wells you will be using for standard transfection by dragging the cursor over the 96 well plate diagram. Use a minimum of 3 wells to pool for each experimental sample.</li>
 <li>Input the program code: in part1 select 'FF' and in part2 select '168' from the pull down menus</li>
 <li>From Solution box select 'Monocyte, human'</li>
 <li>Under Control Option select 'standard'. </li>
 <li>Click on Apply.</li>
 <li>To include a no-transfection control, select further wells from the diagram as required and then choose 'No Program Control' from Control Option and click on Apply. </li>
 <li>Select any remaining unused wells on the plate diagram and click on Undefine. </li>
</ol>
2. Prepare DCs for transfection
<ol>
 <li>All work should be done under sterile conditions in a cell culture hood where possible. Prepare the nucleofection solution by adding 96-well supplement to Human Monocyte 96-well Nucleofector Solution in the ratio of 450 to 2025. Mix and allow to warm to room temperature. You will need 20&mu;l of nucleofection solution per well. Make an excess of 10% to allow for pipetting error.</li>
 <li>Place the number of nucleocuvette modules you need into the nucleocuvette plate in the correct orientation ie inserting the first one in to rows 1 and 2.</li>
 <li>Determine the number of DCs needed for your experiment calculating on 500,000 cells per well and pellet by centrifuging at 400g for 10min. Carefully remove the supernatant.</li>
 <li>Resuspend the cells in the nucleofection solution by gently pipetting up and down a few times.</li>
 <li>Divide the correct volume of resuspended cells into eppendorfs labeled for the particular treatment eg GLO and RIG-I.</li>
 <li>Add 0.25&mu;g siRNA per 500,000 cells and mix by pipetting. Use non-targeting siRNA in your no-transfection control sample.</li>
 <li>Pipette 20&mu;l of the above mixtures into the nucleocuvette modules, according to your experimental layout, ensuring the liquid is delivered to the bottom of the well. </li>
 <li>Cover the nucleocuvette plate with the lid and tap the plate on a hard surface a couple of times to help ensure removal of air bubbles.</li>
</ol>
3. Transfect DCs
<ol>
 <li>Insert plate into the Nucleofector 96-well shuttle tray, click on Upload and then start. </li>
 <li>Follow progress of transfection process on the lap top display; a black cross on a green background signifies a successful transfection in that well, whereas a black bar on a red background means it was unsuccessful.</li>
 <li>On completion of the transfection process, remove the plate and add 80&mu;l of DC growth medium to each well using a multichannel pipette.</li>
 <li>Incubate plate for 10 min at 37&deg;C and 5% CO2.</li>
 <li>Transfer the 100&mu;l volume from the nucleocuvettes into matrix tubes containing 100&mu;l of pre-warmed DC growth medium, maintaining the correct orientation. </li>
 <li>Remove and discard those tubes where transfection did not occur.</li>
 <li>Incubate at 37&deg;C and 5% CO2 for 24h or other desired time interval.</li>
</ol>
4. Infect cells with NDV
<ol>
 <li>Remove matrix tubes from the incubator to the cell culture hood and pool tubes for each experimental sample into eppendorfs</li>
 <li>Pellet the cells gently by spinning in a desk top centrifuge for 5 min and remove supernatant.</li>
 <li>Resuspend the cells in serum-free growth medium containing NDV at an MOI of 1 and incubate at 37&deg;C and 5% CO2 for 45 min, with eppendorfs loosely covered in a sterile fashion.</li>
 <li>Add 900&mu;l of DC growth medium, and re-incubate for 8-10 h.</li>
</ol>
5. Harvest cells
<ol>
 <li>Pellet cells by spinning eppendorfs in a desk top centrifuge and remove supernatant.</li>
 <li>Harvest cells for RNA or protein extraction according to your protocol.</li>
</ol>
6. Representative results:
Using our optimized protocol we transfected DCs with RIGI-targeting siRNA for 24 h and then infected the cells with NDV (a paramyxovirus detected by RIG-I) to stimulate the interferon response pathway. By qRT-PCR analysis we demonstrated knock down of the gene by 75% at the transcription level. We also observed a similar reduction in the expression of IFN&beta;, which is a downstream effector of RIG-I in the IFN signaling cascade. Furthermore, we observed that the expression of IFN&beta; in non-infected, control-transfected cells was not detectable while that of MxA, an IFN&beta; downstream response gene, was minimal (Figure 1A). 
A second transfection of DCs with RIGI-targeting siRNA was performed using enough cells to include Western blot analysis. Where the RT-PCR results were similar to what we saw previously (62% and 66% knock down of RIG-I and IFN&beta; expression respectively) (Figure 1B), the Western blot probed for RIG-I revealed that the expression of this gene had been completely blocked (Figure 1C).
<img src="/files/ftp_upload/2766/2766fig1.jpg" alt="Figure 1A"/> Figure 1. (A) MoDCs were transfected with either siRNA targeting RIG-I or nonspecific GLO siRNA and the effect on the expression of RIG-I, IFN&beta; and MxA determined by qRT-PCR. The transfection protocol used a monocyte-specific buffer and nucleoporation program FF168 (Lonza Walkersville Inc.) Following incubation for 24 h, transfected cells were either infected with NDV (+) or left uninfected (-). After a further incubation for 10 h, cells were harvested and RNA extracted Transcript levels represent the results of two replicate experiments. Taken from Bowles et al 16. 
(B) MoDCs obtained from a different buffy coat as used for Figure 1A were again transfected with either RIG-I-targeting siRNA or nonspecific GLO siRNA using the same transfection protocol as above. An additional control using untransfected MoDCs was incorporated into the experiment. Following a 24 h incubation all cells were infected with NDV and incubated for a further 10 h before being harvested for both RNA and protein extraction. Transcript levels of RIG-I, IFN&beta; and MxA as determined by qRT-PCR represent the results of two replicate experiments. Taken from Bowles et al 16.
(C) Lysates from cells described in Figure 1B above were analyzed by Western blot. Lanes 1 and 2, lysates from untransfected cells; lanes 3 and 4, lysates from GLO siRNA transfected cells; lanes 5 and 6, lysates from cells transfected with RIG-I-targeting siRNA. Samples were probed for RIG-I and also GAPDH (as a loading control) and were run in duplicate. Taken from Bowles et al 16.

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+dendritic+cells+in+the+innate+immune+system.">The role of dendritic cells in the innate immune system.</a> Microbes Infect. 2, 257-272 (2000).</li><li>Hamm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfection+method+for+primary+cells.">Efficient transfection method for primary cells.</a> Tissue Eng. 8, 235-245 (2002).</li><li>Henderson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dendritic+cells+genetically+engineered+to+express+high+levels+of+the+human+epithelial+tumor+antigen+mucin+(MUC-1).">Human dendritic cells genetically engineered to express high levels of the human epithelial tumor antigen mucin (MUC-1).</a> Cancer Res. 56, 3763-3770 (1996).</li><li>Reeves, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+transduction+of+human+dendritic+cells+with+a+tumor-associated+antigen+gene.">Retroviral transduction of human dendritic cells with a tumor-associated antigen gene.</a> Cancer Res. 56, 5672-5677 (1996).</li><li>Aicher, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+retroviral+mediated+transduction+of+a+reporter+gene+in+human+dendritic+cells:+feasibility+of+therapy+with+gene-modified+antigen+presenting+cells.">Successful retroviral mediated transduction of a reporter gene in human dendritic cells: feasibility of therapy with gene-modified antigen presenting cells.</a> Exp. Hematol. 25, 39-44 (1997).</li><li>Thomas, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+problems+with+the+use+of+viral+vectors+for+gene+therapy.">Progress and problems with the use of viral vectors for gene therapy.</a> Nat Rev Genet. 4, 346-358 (2003).</li><li>Jooss, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+dendritic+cells+by+DNA+viral+vectors+directs+the+immune+response+to+transgene+products+in+muscle+fibers.">Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers.</a> J. Virol. 72, 4212-4223 (1998).</li><li>Mitchell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-transfected+dendritic+cells+in+cancer+immunotherapy.">RNA-transfected dendritic cells in cancer immunotherapy.</a> J. Clin. Invest. 106, 1065-1069 (2000).</li><li>Mincheff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+transfection+and/or+cross-priming+of+dendritic+cells+following+DNA+and+adenoviral+immunizations+for+immunotherapy+of+cancer-changes+in+peripheral+mononuclear+subsets+and+intracellular+IL-4+and+IFN-gamma+lymphokine+profile.">In vivo transfection and/or cross-priming of dendritic cells following DNA and adenoviral immunizations for immunotherapy of cancer-changes in peripheral mononuclear subsets and intracellular IL-4 and IFN-gamma lymphokine profile.</a> Crit. Rev. Oncol. Hematol. 39, 125-132 (2001).</li><li>Roth, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helper-dependent+adenoviral+vectors+efficiently+express+transgenes+in+human+dendritic+cells+but+still+stimulate+antiviral+immune+responses.">Helper-dependent adenoviral vectors efficiently express transgenes in human dendritic cells but still stimulate antiviral immune responses.</a> J. Immunol. 169, 4651-4656 (2002).</li><li>Tendeloo, V. F. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+gene+delivery+by+mRNA+electroporation+in+human+hematopoietic+cells:+superiority+to+lipofection+and+passive+pulsing+of+mRNA+and+to+electroporation+of+plasmid+cDNA+for+tumor+antigen+loading+of+dendritic+cells.">Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells.</a> Blood. 98, 49-56 (2001).</li><li>Lenz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoporation+of+dendritic+cells:+efficient+gene+transfer+by+electroporation+into+human+monocyte-derived+dendritic+cells.">Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte-derived dendritic cells.</a> FEBS Lett. 538, 149-154 (2003).</li><li>Prechtel, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+interfering+RNA+(siRNA)+delivery+into+monocyte-derived+dendritic+cells+by+electroporation.">Small interfering RNA (siRNA) delivery into monocyte-derived dendritic cells by electroporation.</a> J. Immunol. Methods. 311, 139-152 (2006).</li><li>Bowles, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+efficient+high-throughput+plasmid+and+siRNA+transfection+of+human+monocyte-derived+dendritic+cells+without+cell+maturation.">Validation of efficient high-throughput plasmid and siRNA transfection of human monocyte-derived dendritic cells without cell maturation.</a> J. Immunol. 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No conflicts of interest declared.

Efficient transfection of na&iuml;ve primary dendritic cells is important for high throughput analysis and reverse engineering of cellular inflammatory pathways in this key cell mediating the innate-adaptive immune transition. However, most investigators find that these cells are difficult to transfect both efficiently and without the transfection procedure inducing cell maturation when using standard transfection techniques. We investigated whether these limitations could be overcome by high throughput protocol optimiza...

<table border="1">
	<tbody>
		<tr>
			<th>Equipment/Reagent</th>
			<th>Company</th>
			<th>Catalogue #</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Amaxa Nucleofector 96-well Shuttle</td>
			<td>Lonza</td>
			<td>108S0109</td>
			<td>Serial number</td>
		</tr>
		<tr>
			<td>Amaxa Human Monocyte 96-well Nucleofector Kit</td>
			<td>Lonza</td>
			<td>VHPA-2007</td>
			<td>Contains the Human Monocyte 96-well Nucleofector Solution, the 96-well Supplement and the Nucleocuvettes and plates</td>
		</tr>
		<tr>
			<td>RIG-I siRNA</td>
			<td>Dharmacon</td>
			<td>L-012511-00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GLO siRNA</td>
			<td>Dharmacon</td>
			<td>D-001600-01-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Invitrogen</td>
			<td>11875</td>
			<td>Supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 &mu;g/ml streptomycin to make DC growth medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030081</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15070063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>HyClone</td>
			<td>3070.03</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dendritic Cells</td>
			<td>New York Blood center</td>
			<td>&nbsp;</td>
			<td>DCs are purified from buffy coats using a standard procedure</td>
		</tr>
		
	</tbody>
</table>

optimized protocol for efficient transfection of dendritic cells without cell maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

Watch this Scientific Journal Video about Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation at JoVE.com

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

optimized-protocol-for-efficient-transfection-dendritic-cells-without

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JoVE Journal

Immunology and Infection

20.2K Views.  Mount Sinai School of Medicine. We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Video: Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...