1. Transduction of murine and human primary NK cells with lentivirus
<ol>
	<li>Suspend mouse or human primary NK cells in a 24-well plate at 0.5 &#215; 105/mL of medium in the presence of green fluorescent protein (GFP) lentivirus supernatant at 5, 10, and 20 multiplicity of infection (MOI) and in the presence of Pb (8 &#181;g/mL), PS (8 &#181;g/mL) or dextran (8 &#181;g/mL).</li>
	<li>Centrifuge the plates at 1,000 &#215; g for 60 min.</li>
	<li>Without decanting the supernatant, culture the cells overnight (16-18 h) in a 37 &#176;C incubator infused with 5.2% CO2.</li>
	<li>Wash with 10 mL of PBS and resuspend in 2 mL of complete Roswell Park Memorial Institute (RPMI) 1640 culture medium in the presence of interleukin 2 (IL-2, 300 U/mL).</li>
	<li>Harvest the transduced NK cells on day 7 by gently tapping the flasks.
	<ol>
		<li>Activate the harvested cells with titrated concentrations of plate-bound anti-natural killer group 2D (NKG2D) (A10) mAb by coating 96-well, highly protein-absorbent polystyrene plates with 2.5 &#181;g/mL concentrations of anti-NKG2D mAb overnight.</li>
		<li>Wash each well with 100 &#181;L of phosphate-buffered saline (PBS) three times before the addition of primary NK cells.</li>
	</ol>
	</li>
	<li>Collect culture supernatants using a multi-channel pipette between 16 and 18 h post-activation to quantify cytokines such as interferon-gamma (IFN-&#947;). Generate standard curves using the recombinant cytokines provided with enzyme-linked immunosorbent assay (ELISA) kits.</li>
	<li>Test the NK cell viability using annexin-V/7-amino-actinomycin D (7-AAD) staining and determine the percent of necrotic cells among the transformed NK cells using a flow cytometer.
	<ol>
		<li>To perform this assay, harvest the murine and human NK cells four days after transduction, wash two times with 10 mL of cold PBS at 500 &#215; g for 5 min each, and incubate with an Annexin-V (PE)/7-AAD kit.</li>
	</ol>
	</li>
	<li>Use appropriate flow cytometry software to analyze the data. Select the live-cell population and analyze the expression of GFP (fluorescein isothiocyanate [FITC] channel) as a measure of viral transduction.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>90-64-91-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene (Pb)</td>
			<td>Sigma-Aldrich</td>
			<td>TR-1003</td>
			<td></td>
		</tr>
		<tr>
			<td>Protamine sulfate (PS)</td>
			<td>Sigma-Aldrich</td>
			<td>p3369</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Interferon gamma (IFN-&#947; )</td>
			<td>eBioscience</td>
			<td>14-7311-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium lodide staining solution</td>
			<td>BD</td>
			<td>51-66211E</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Thermo Fisher</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>PHOENIX</td>
			<td>NDC 57319-559-05</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>Corning</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA kits</td>
			<td>Ebioscience</td>
			<td>00-4201-56</td>
			<td></td>
		</tr>
	</tbody>
</table>

enhancing viral transduction in natural killer cells using a cationic polymer

Source: Nanbakhsh, A., et al. <a href='https://app.jove.com/v/55063'>Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells.</a> J. Vis. Exp. (2018).
This video demonstrates an assay for cationic polymer-mediated viral transduction in natural killer (NK) cells. The cells are incubated with transgenic lentivirus vectors encoding a fluorescent protein. The addition of a cationic polymer enables the virus to attach to the cells and integrate its RNA into the host genome, expressing the fluorescent protein. The fluorescence is quantified to assess the polymer-induced enhancement of transduction.

Enhancing Viral Transduction in Natural Killer Cells Using a Cationic Polymer

1. Transduction of murine and human primary NK cells with lentivirus

	Suspend mouse or human primary NK cells in a 24-well plate at 0.5 &#215; 105/mL of ...

Take natural killer, or NK, cells. Add lentivirus vectors comprising viral enzymes and engineered RNA molecules that encode a fluorescent protein.
Add cationic polymers and sediment the cells and viruses.
The polymers neutralize the negative charges on both the virus and the cell, facilitating membrane fusion.
The RNA is reverse-transcribed into DNA and integrated into the host genome, enabling fluorescent protein expression.
Add media supplemented with the cytokine interleukin-2, or IL-2.
IL-2 triggers intracellular signaling that promotes NK cell survival and proliferation while preserving transgene expression.
Transfer the mixture to a well coated with antibodies that bind to specific immunoreceptors on NK cells, triggering them to release antiviral cytokines.
Quantify the released cytokines to assess the polymer's impact on cytokine production.
Add fluorescent dyes to label the DNA of necrotic cells.
Using flow cytometry, identify live cells and quantify the fluorescent protein's signal to measure the efficiency of polymer-induced transduction.

Suspend mouse or human primary NK cells in the wells of a 24-well plate at 0.5 x 105 per milliliter of medium with GFP lentivirus supernatant at an MOY of 5, 10, and 20 and in the presence of polybrene, protamine sulfate, or dextran.
Centrifuge the plates at 1,000 times g for 60 minutes. Then, without decanting the supernatant, culture the cells in a 37 degrees Celsius incubator infused with 5.2% carbon dioxide overnight. After the cells have been washed with 10 milliliters of PBS, use 2 milliliters of RPMI 1640 medium with IL-2 to resuspend the cells.
The day before harvesting the cells, coat 96-well highly protein absorbent polystyrene plates with 2.5 micrograms per milliliter of anti-NKG2D monoclonal antibodies, and incubate the plates at room temperature or in the fridge overnight. The following day, use 100 microliters of PBS to wash each well three times.
Harvest the transduced NK cells by first gently tapping the plates. Then add 100 microliters of the cells to each well of the 96-well plates. 16 to 18 hours post-activation, use a multichannel pipette to collect the supernatants. Then, using the recombinant cytokines from an ELISA kit, generate a standard curve and quantify the cytokines such as interferon-gamma in the supernatant.
To test the NK cell viability, four days after transduction, wash the cells with 10 milliliters of cold PBS two times. Then, harvest the cells and use an annexin-V/7-amino-actinomycin D to stain them. Use flow cytometry to determine the percent of necrotic cells among the transformed NK cells, and analyze the data according to the text protocol.

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832 Views. Take natural killer, or NK, cells. Add lentivirus vectors comprising viral enzymes and engineered RNA molecules that encode a fluorescent protein. Add cationic polymers and sediment the cells and viruses. The polymers neutralize the negative charges on both the virus and the cell, facilitating membrane fusion. The RNA is reverse-transcribed into DNA and integrated into the host genome, enabling fluorescent protein expression. Add media supplemented with the cytokin...

Enhancing Viral Transduction in Natural Killer Cells Using a Cationic Polymer

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Name	Company	Catalog Number
Dextran	Sigma-Aldrich	90-64-91-9
Polybrene (Pb)	Sigma-Aldrich	TR-1003
Protamine sulfate (PS)	Sigma-Aldrich	p3369
RPMI1640	Corning	10-040-CV
Interferon gamma (IFN-γ )	eBioscience	14-7311-85
Propidium lodide staining solution	BD	51-66211E
Lipofectamine 3000	Thermo Fisher	L3000015
Isoflurane	PHOENIX	NDC 57319-559-05
293T cells	ATCC	CRL-3216
T75 flasks	Corning	430641U
ELISA kits	Ebioscience	00-4201-56