This work was supported by the US National Institutes of Health grants (R01HL109102, P01HL107202, U19CA179512, F31HL131361), a Leukemia &amp; Lymphoma Society scholar award (K.M.A.), and the National Institute of General Medical Sciences (NIGMS) Medical Scientist Training Program (Grant #T32GM007618) (M.M.).

This protocol adheres to UCSF&#39;s guidelines for human research ethics.
1. Preparation of T Cell Culture, Isolation of CD4+ T Cells, and Th17 Polarization
<ol>
	<li>On Day 0, coat 6-well tissue culture plates with 1.5 mL per well of anti-human CD3 (2 &#181;g/mL) and anti-human CD28 (4 &#181;g/mL) in PBS with calcium and magnesium for at least 2 h at 37 &#176;C.
	<ol>
		<li>Alternatively, coat the plates overnight at 4 &#176;C. Wrap plates in parafilm.</li>
	</ol>
	</li>
	<li>Prepare the cord blood mononuclear cells (CBMCs) by density gradient centrifugation per manufacturer&#39;s instructions. 
	CAUTION: Work carefully and ensure proper personal protective equipment (PPE) is worn when handling human blood to avoid any risk of exposure to blood-borne pathogens.</li>
	<li>Once the mononuclear cells have been isolated and washed, perform human CD4+ T cell isolation by negative selection using a commercial Human CD4+ T cell kit. 
	NOTE: If there is red blood cell contamination after mononuclear cell isolation, an optional red blood cell lysis may be performed prior to the CD4+ T cell isolation steps. Resuspend the mononuclear cells in 1 mL of isolation buffer (2% FBS in PBS). Add 5 mL of 1x lysing solution. Incubate for 15 min at room temperature. Then add 5 mL of isolation buffer and centrifuge at 300 x g for 5 min at 4 &#176;C to pellet the cells.
	<ol>
		<li>Resuspend the mononuclear cells at a density of 50 x 106 per 500 &#181;L in the isolation buffer in a new 5 mL tube.</li>
		<li>Add 100 &#181;L of FBS and 100 &#181;L of the Antibody Mix per tube then incubate each tube at 4 &#176;C for 20 min on an orbital shaker to mix well.</li>
		<li>After the incubation, add 3-4 mL of isolation buffer to wash the cells. Centrifuge the cells at 300 x g for 8 min at 4 &#176;C to pellet the cells. Carefully aspirate the supernatant.</li>
		<li>During this spin, pre-wash the magnetic beads. Transfer the desired amount of magnetic beads into a new tube (used at 1:1 with the cells) and add an equal volume of isolation buffer to wash the beads. Mix well&#160;using a 1 mL micropipette and then place the tube in the magnet for at least 1 min. Carefully aspirate the supernatant. Resuspend the magnetic beads in the same volume initially transferred prior to the wash.</li>
		<li>Resuspend the cells in each tube with 500 &#181;L of the isolation buffer and add 500 &#181;L of pre-washed magnetic beads.</li>
		<li>Incubate the cells with the magnetic beads for 15 min on an orbital shaker at room temperature (18 &#176;C to 25 &#176;C) to mix well.</li>
		<li>After the incubation, thoroughly pipet the cells using a 1 mL micropipette at least 10 times. Then add 3-4 mL of isolation buffer and place each tube in the magnet for 2 min.</li>
		<li>Carefully transfer the negatively selected CD4+ T cells that are in the supernatant to a new tube.</li>
	</ol>
	</li>
	<li>Once CD4+ T cell isolation is completed, count the cells with a hemocytometer and keep the cells on ice.</li>
	<li>Wash the antibody-coated plates two times with PBS. Then add 1.5 mL of 2x mix of Th17-polarizing media to each well: anti-human IFN&#947; (20 &#181;g/mL), anti-human IL-4 (20 &#181;g/mL), human TGF&#946; (10 ng/mL), human IL-1&#946; (40 ng/mL), human IL-23 (40 ng/mL), human IL-6 (50 ng/mL) all diluted in a serum-free base media (supplemented with&#160;2 mM L-glutamine, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and 100 &#181;M 2-mercaptoethanol). 
	NOTE: Transfection and RNA interference does work in serum-containing media. The purpose of using serum-free media with this protocol is to achieve better IL-17A production.</li>
	<li>Centrifuge the human CD4+ T cells at 500 x g for 5 min at 4 &#176;C to pellet cells. Carefully aspirate the supernatant. Then resuspend the cells in serum-free base media and plate the cells at a density of 3 x 106 in 1.5 mL per well so that the final volume is 3 mL per well and polarizing cytokines are now at a 1x final concentration.
	<ol>
		<li>Place the plates in 5% CO2, 37 &#176;C incubator for two days.</li>
	</ol>
	</li>
</ol>
2. Electroporation of In Vitro Polarized Human Th17 Cells
<ol>
	<li>On Day 2, coat 48-well tissue culture plates with 250 &#181;L per well of anti-human CD3 (2 &#181;g/mL) and anti-human CD28 (4 &#181;g/mL) in PBS with calcium and magnesium for at least 2 h at 37 &#176;C.
	<ol>
		<li>Alternatively, coat the plates overnight at 4 &#176;C. Wrap plates in parafilm.</li>
	</ol>
	</li>
	<li>Prepare small RNAs for transfection. For each transfection, aliquot 1 &#181;L of a 5 &#181;M stock solution of siRNA into a 1.5 mL microcentrifuge tube. Include appropriate chemistry-matched small RNA control. Keep all tubes on ice.</li>
	<li>Wash the antibody-coated plates two times with PBS. Then add 500 &#181;L of 1X Th17-polarizing media to each well: anti-human IFN&#947; (10 &#181;g/mL), anti-human IL-4 (10 &#181;g/mL), human TGF&#946; (5 ng/mL), human IL-1&#946; (20 ng/mL), human IL-23 (20 ng/mL), human IL-6 (25 ng/mL) all diluted in a serum-free base media (supplemented with&#160;2 mM L-glutamine, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and 100 &#181;M 2-mercaptoethanol).</li>
	<li>After the culture plates and transfection reagents are prepared, resuspend the cells with a 1 mL micropipette, pipetting gently but ensuring that all the cells are detached from the bottom of the wells. Pool the cells into a conical tube and centrifuge at 500 x g for 5 min at 4 &#176;C. 
	NOTE: For culture periods longer than the four-day protocol presented herein, the cells should be transfected approximately every 3 days using this same protocol. Step 2.4 should be modified however since the cells treated with different small RNAs should not be pooled. All of the conditions being tested must be collected, counted, and transfected separately.</li>
	<li>Carefully aspirate the supernatant, and then resuspend the cells in at least 1 mL of PBS to wash. Count the live Th17 cells (e.g. with a hemocytometer using Trypan Blue exclusion to assess viability) and then transfer the cells to a microcentrifuge tube.</li>
	<li>Centrifuge at 500 x g for 5 min at room temperature to pellet the cells.</li>
	<li>Carefully aspirate the supernatant, and then resuspend the cells using the provided resuspension buffer from the transfection system 10 &#181;L kit at a density of 2.5-4 x 107 cells per mL. Keep cells at room temperature. 
	NOTE: As a guideline, try to prepare only as many cells as can be transfected within 30 min. Multiple batches can be compared.</li>
	<li>Add 9 &#181;L of cells to 1 &#181;L of small RNA in each microcentrifuge tube. Pipet once to mix the cells and small RNAs for transfection then load into the provided pipette electrode tip. 
	NOTE: Typically, add 9.5 &#181;L of cell suspension to ensure there is enough of the mixture to prevent creating bubbles in the pipet electrode tip prior to transfection.</li>
	<li>Fill the provided cuvette with 3 mL of room temperature Electrolytic Buffer E. Place the cuvette inside the pipette station and then place the pipette into position inside the cuvette.</li>
	<li>Immediately electroporate each 10 &#181;L mix of cells and small RNA using the following parameters: pulse voltage 1,500-1,550 V, pulse width 10 ms, and 3 pulses total on the transfection device.</li>
	<li>After the electroporation is complete, directly add the cell mixture to 500 &#181;L of 1X Th17-polarizing media in prepared wells of a culture plate. Place plates in a 5% CO2, 37 &#176;C incubator for two more days. 
	NOTE: Wash the pipette electrode tip by pipetting up and down in PBS in between each transfection.</li>
</ol>
3. Harvesting Human Th17 Cells
<ol>
	<li>Resuspend the cells in culture media with a micropipette, pipetting gently but ensuring that all the cells are detached from the bottom of the wells. Prepare the cells for functional and/or gene expression assays. 
	NOTE: Routinely, enough cells are yielded from a single well of transfected Th17 cells to be used for flow cytometric analysis of surface marker and intracellular cytokine and transcription factor staining or for preparation of RNA for gene expression analysis.</li>
</ol>

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	<li>Zhu, J., Yamane, H., Paul, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+Effector+CD4+T+Cell+Populations*.">Differentiation of Effector CD4 T Cell Populations*.</a> Ann Rev Immunol. 28 (1), 445-489 (2010).</li><li>Korn, T., Bettelli, E., Oukka, M., Kuchroo, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17+and+Th17+Cells.">IL-17 and Th17 Cells.</a> Ann Rev Immunol. 27 (1), 485-517 (2009).</li><li>Gaffen, S. L., Jain, R., Garg, A. V., Cua, D. 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+IL-23-IL-17+immune+axis:+from+mechanisms+to+therapeutic+testing.">The IL-23-IL-17 immune axis: from mechanisms to therapeutic testing.</a> Nature. 14 (9), 585-600 (2014).</li><li>Romagnani, S., Maggi, E., Liotta, F., Cosmi, L., Annunziato, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+and+origin+of+human+Th17+cells.">Properties and origin of human Th17 cells.</a> Mol Immunol. 47 (1), 3-7 (2009).</li><li>Acosta-Rodriguez, E. V., 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=Surface+phenotype+and+antigenic+specificity+of+human+interleukin+17-producing+T+helper+memory+cells.">Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells.</a> Nature Immunol. 8 (6), 639-646 (2007).</li><li>Annunziato, F., 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=Phenotypic+and+functional+features+of+human+Th17+cells.">Phenotypic and functional features of human Th17 cells.</a> J Exp Med. 204 (8), 1849-1861 (2007).</li><li>Du, C., 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=MicroRNA+miR-326+regulates+TH-17+differentiation+and+is+associated+with+the+pathogenesis+of+multiple+sclerosis.">MicroRNA miR-326 regulates TH-17 differentiation and is associated with the pathogenesis of multiple sclerosis.</a> Nature Immunol. 10 (12), 1252-1259 (2009).</li><li>Escobar, T. M., 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=miR-155+Activates+Cytokine+Gene+Expression+in+Th17+Cells+by+Regulating+the+DNA-Binding+Protein+Jarid2+to+Relieve+Polycomb-Mediated+Repression.">miR-155 Activates Cytokine Gene Expression in Th17 Cells by Regulating the DNA-Binding Protein Jarid2 to Relieve Polycomb-Mediated Repression.</a> Immunity. 40 (6), 865-879 (2014).</li><li>Wang, 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=Negative+regulation+of+Hif1a+expression+and+TH17+differentiation+by+the+hypoxia-regulated+microRNA+miR-210.">Negative regulation of Hif1a expression and TH17 differentiation by the hypoxia-regulated microRNA miR-210.</a> Nature Immunol. 15 (4), 393-401 (2014).</li><li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Anal bioanal chem. 397 (8), 3173-3178 (2010).</li><li>Steiner, D. F., 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=MicroRNA-29+Regulates+T-Box+Transcription+Factors+and+Interferon-&amp;gamma;+Production+in+Helper+T+Cells.">MicroRNA-29 Regulates T-Box Transcription Factors and Interferon-&amp;gamma; Production in Helper T Cells.</a> Immunity. 35 (2), 169-181 (2011).</li><li>Simpson, L. J., 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=A+microRNA+upregulated+in+asthma+airway+T+cells+promotes+TH2+cytokine+production.">A microRNA upregulated in asthma airway T cells promotes TH2 cytokine production.</a> Nature Immunol. 15 (12), 1162-1170 (2014).</li><li>Pua, H. 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=MicroRNAs+24+and+27+Suppress+Allergic+Inflammation+and+Target+a+Network+of+Regulators+of+T+Helper+2+Cell-Associated+Cytokine+Production.">MicroRNAs 24 and 27 Suppress Allergic Inflammation and Target a Network of Regulators of T Helper 2 Cell-Associated Cytokine Production.</a> Immunity. 44 (4), 821-832 (2016).</li><li>Flaherty, S., Reynolds, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Na&#239;ve+CD4++T+Cell+Isolation+and+In+vitro+Differentiation+into+T+Cell+Subsets.">Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets.</a> J Vis Exp. (98), (2015).</li><li>Schumann, K., 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=Generation+of+knock-in+primary+human+T+cells+using+Cas9+ribonucleoproteins.">Generation of knock-in primary human T cells using Cas9 ribonucleoproteins.</a> Proc Natl Acad Sci USA. 112 (33), 10437-10442 (2015).</li></ol>

The authors have nothing to disclose.

This protocol provides an improved method for the delivery of small RNAs into human Th17 cells. Although human Th17 cells were used here, this method of electroporation with small RNAs can be used with other primary human T helper subsets, such as Th1, Th2, and Tregs. It has not worked well for na&#239;ve CD4+ T cells so the cells must be activated in culture prior to transfection. For this protocol, we first optimized the in vitro culture system for better IL-17A production. The biggest factor was ba...

CD4+ T cells are crucial orchestrators of the adaptive immune response. Na&#239;ve CD4+ T cells are capable of developing into several different effector T cells (e.g. Th1, Th2, Th17, etc.), each with their own set of characteristic cytokines and transcription factors, depending upon the local microenvironment1. The lineage decisions that T cells make are critical for both protective immunity and tolerance to self. Th17 cells are one subset of T cells known to combat extracellular bacteria and fungi, but their improper responses are also implicated in the pathogenesis of multiple autoimmune and inflammatory diseases such as multiple sclerosis and psoriasis2,3. Human Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by providing them with an appropriate polarizing environment4. Various combinations of the cytokines IL-1&#946;, IL-23, TGF&#946;, and IL-6 have been used for the development of human Th17 cells. Human Th17 cells express CCR6, a chemokine receptor that is commonly used to identify this cell population and are defined by the expression of their principal transcription factor, ROR&#947;t (encoded by RORC)5,6. Th17 cells have the ability to express multiple cytokines, but IL-17A is the lineage-defining effector cytokine produced by these cells. We examined the expression of all three Th17-associated markers (CCR6, ROR&#947;t, IL-17A) to assess the robustness of our human Th17 in vitro differentiation assay. Additionally, we cultured human CD4+ T cells under non-polarizing conditions, where no cytokines or blocking antibodies were added to the culture media to use as a negative control since expression of these Th17 markers should be very low or absent.
One way to study normal human T cell development and biology is to manipulate gene expression during their development. Short-interfering RNA (siRNA) are synthetic small RNA molecules that target protein-coding mRNAs and can be utilized to reduce specific gene expression. MicroRNAs (miRNAs) are endogenous non-coding small RNAs known to modulate gene expression post-transcriptionally. miRNAs have been shown to play an important role in both murine and human T cell biology, including in Th17 cells7,8,9. It is crucial to have reliable methods of manipulating small RNA activity in human T cells to study their effects on gene expression and ultimately on human T cell biology. Here, we describe an easy-to-use, consistent and reliable protocol that we developed for introducing small synthetic RNAs and locked nucleic acids (LNAs, chemically modified nucleic acids with increased stability) into immune cells, and specifically into human Th17 cells.
There are several alternative methods of introducing small RNAs into mammalian cells, which generally fall into chemical, biological, or physical categories10. Commonly used chemical methods, including lipid-based transfections and calcium-phosphate transfections, rely on creating chemical-DNA complexes that are more efficiently taken up by cells. In general, chemical methods are not as efficient for the transfection of primary T cells. The most common biological method is to use a viral vector (e.g. retrovirus or lentivirus), which directly inserts foreign RNA into a host as a part of its natural replication cycle. Viral transduction typically takes longer to complete, especially when one factors in time for molecular cloning of proviral plasmids. Additionally, viral transduction vectors can be potentially harmful to human researchers. Electroporation is a physical method of inducing membrane permeabilization by subjecting cells to high voltage pulses, allowing nucleic acids to transiently enter into the cell where they can act on their target. Traditional electroporation instruments were not effective for transfecting primary lymphocytes. However, optimized next generation electroporation has proven to be capable of transfecting T cells at very high efficiency, especially when the material to be transfected is small RNA. The term next generation is loosely used to differentiate the two newer platforms (e.g., Neon, Amaxa) from traditional electroporation machines. Additionally, this method is easily scalable for moderate throughput screens with up to approximately 120 small RNAs in a single experiment, often using validated synthetic reagents. Importantly, successful transfections can be achieved in as little as 16 h after T cell activation. The disadvantage of this method, however, is that it does not result in stable genomic incorporation, and is therefore transient. Hence, it is worth the extra effort to create a stable expression construct that can be packaged into a viral vector and successfully expressed in T cells in cases where long-term expression of a small RNA is required.
We have used a next generation transfection (e.g., Neon) to deliver diverse synthetic single or double-stranded RNA or LNA oligonucleotide tools for different purposes11,12,13. Efficient RNA interference can be induced in primary mouse and human T cells using double-stranded short-interfering RNA (siRNA). This protocol describes optimized conditions for using this technique in human Th17 cells. In addition to siRNAs, commercially available synthetic miRNA mimics and inhibitors can be used to study miRNA gain and loss of function. miRNA mimics are double-stranded RNA molecules very similar to siRNAs, but designed with the sequence of endogenous mature miRNAs. miRNA inhibitors are chemically-modified RNA and/or LNA based single stranded oligonucleotides that bind to native miRNAs and antagonize their function. We have found that all of these tools can be used effectively in cultured primary T lymphocytes, including but not limited to human Th17 cells.

<table><tbody><tr><td>anti-human IL-17A PE</td><td>ebioscience</td><td>12-7179-42&amp;nbsp;</td><td>Clone: eBio64DEC17</td></tr><tr><td>anti-human IFNg FITC</td><td>ebioscience</td><td>11-7319-82</td><td>Clone: 4S.B3</td></tr><tr><td>anti-human CD4 eVolve605</td><td>ebioscience</td><td>83-0047-42</td><td>Clone: SK3</td></tr><tr><td>mouse anti-human CD196 (CCR6) BV421</td><td>BD biosciences</td><td>562515</td><td>Clone: 11A9</td></tr><tr><td>anti-human RORgt AF647</td><td>BD biosciences</td><td>563620</td><td>Clone: Q21-559</td></tr><tr><td>anti-human CD45 eFluor450</td><td>ebioscience</td><td>48-9459-42</td><td>Clone: 2D1</td></tr><tr><td>Foxp3/Transcription Factor Staining Buffer Set</td><td>ebioscience</td><td>00-5523-00</td><td>For intracellular transcription factor flow cytometry staining</td></tr><tr><td>Hu FcR Binding Inhibitor Purified</td><td>ebioscience</td><td>14-9161-71</td><td></td></tr><tr><td>ImmunoCult-XF T Cell Expansion Medium</td><td>Stemcell Technologies</td><td>10981</td><td>&quot;Serum-Free Base Media&quot;</td></tr><tr><td>MACS CD28 pure functional grade, human</td><td>Miltenyi Biotec</td><td>130-093-375</td><td>Clone: 15E8</td></tr><tr><td>anti-human CD3 Purified</td><td>UCSF monoclonal antibody core</td><td>N/A</td><td>Clone: OKT-3</td></tr><tr><td>LEAF purified anti-human IL-4</td><td>Biolegend</td><td>500815</td><td>Clone: MP4-25D2</td></tr><tr><td>anti-human IFNg, functional grade purified</td><td>ebioscience</td><td>16-7318-85</td><td>Clone: NIB42</td></tr><tr><td>Recombinant human IL-23</td><td>Peprotech&amp;nbsp;</td><td>200-23</td><td></td></tr><tr><td>Recombinant human IL-1&amp;beta;</td><td>Peprotech&amp;nbsp;</td><td>200-01B</td><td></td></tr><tr><td>Recombinant human TGF-&amp;beta;1</td><td>Peprotech&amp;nbsp;</td><td>100-21C</td><td></td></tr><tr><td>Recombinant human IL-6</td><td>Peprotech&amp;nbsp;</td><td>200-06</td><td></td></tr><tr><td>siGENOME Control Pool, Non-targeting #2</td><td>Dharmacon</td><td>D-001206-14-05</td><td></td></tr><tr><td>siGENOME SMARTpool Human RORC</td><td>Dharmacon</td><td>M-003442-00</td><td></td></tr><tr><td>siGENOME SMARTpool Human PTPRC</td><td>Dharmacon</td><td>M-008067-01</td><td></td></tr><tr><td>Dynabeads Untouched Human CD4 T Cells Kit</td><td>ThermoFisher Scientific</td><td>11346D</td><td>Human CD4&lt;sup&gt;+&lt;/sup&gt; T cell Isolation Kit</td></tr><tr><td>Neon Tranfection System</td><td>ThermoFisher Scientific</td><td>MPK5000</td><td>Next generation electroporation instrument</td></tr><tr><td>Neon Tranfection System 10 &amp;mu;L Kit</td><td>ThermoFisher Scientific</td><td>MPK1096</td><td></td></tr><tr><td>Resuspension Buffer T</td><td>ThermoFisher Scientific</td><td>Provided in kit (MPK1096)</td><td>&quot;Transfection Resuspension Buffer&quot;</td></tr><tr><td>Lymphoprep</td><td>Stemcell Technologies</td><td>#07801</td><td>Density Gradient Medium</td></tr><tr><td>costar 6-well tissue culture treated plates</td><td>Corning</td><td>3516</td><td>flat bottom plates</td></tr><tr><td>costar 48-well tissue culture treated plates</td><td>Corning</td><td>3548</td><td>flat bottom plates</td></tr><tr><td>BD Pharm Lyse lysing buffer, 10x</td><td>BD biosciences</td><td>555899</td><td>Must make 1x solution with distilled water prior to use</td></tr></tbody></table>

small rna transfection in primary human th17 cells by next generation electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

The first step to developing a reliable system of successfully electroporating human Th17 cells was to generate robust in vitro differentiated human Th17 cell cultures. T cells cultured under Th17-polarizing conditions expressed the chemokine receptor CCR6 and the transcription factor ROR&#947;t (Figure 1A, left). These markers were not expressed when T cells were cultured under non-polarizing (ThN) conditions (Figure 1A, right). T cells cultured...

Watch this Scientific Journal Video about Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation at JoVE.com

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

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Immunology and Infection

7.8K Views.  University of California, San Francisco. Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

Video: Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

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.

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 ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Bioengineering

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Biology

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. ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

Medicine

Th17 Cell Differentiation from Naive CD4+ T-Cells Using Flow Cytometry

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

Author Spotlight: Achieving High-Purity In Vitro Differentiation of Th17 Cells Using Cytokine Concentration Modulation

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

Retroviral Transduction of Guide RNA for Gene Editing in Th17 Differentiation

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining na&#239;ve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate na&#239;ve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells ...

Engineering and Expansion of Human CAR Tregs for Immune Regulation Therapies

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including their interplay with significant human pathogens such as the human immunodeficiency virus (HIV). Compared to other immortalized cell lines of myeloid origin, THP-1 cells retain many intact inflammatory signaling pathways and display phenotypic characteristics that more closely resemble those of primary monocytes, including the ability to differentiate into macrophages when treated with phorbol-12-myristate 13-acetate (PMA). The use of CRISPR-Cas9 technology to engineer THP-1 cells through targeted gene knockout (KO) provides a powerful approach to better characterize immune-related mechanisms, including virus-host interactions. This article describes a protocol for efficient CRISPR-Cas9-based engineering using electroporation to deliver pre-assembled Cas9:sgRNA ribonucleoproteins into the cell nucleus. Using multiple sgRNAs targeting the same locus at slightly different positions results in the deletion of large DNA fragments, thereby increasing editing efficiency, as assessed by the T7 endonuclease I assay. CRISPR-Cas9-mediated editing at the genetic level was validated by Sanger sequencing followed by Inference of CRISPR Edits (ICE) analysis. Protein depletion was confirmed by immunoblotting coupled with a functional assay. Using this protocol, up to 100% indels in the targeted locus and a decrease of over 95% in protein expression were achieved. The high editing efficiency makes it convenient to isolate single-cell clones by limiting dilution.

The THP-1 cell line is widely used as a model to investigate the functions of human monocytes/macrophages across various biology-related research areas. This article describes a protocol for efficient CRISPR-Cas9-based engineering and single-cell clone isolation, enabling the production of robust and reproducible phenotypic data.

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

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