This work was supported by the National Heart Lung and Blood Institute of the National Institutes of Health under grant R00HL130456, the National Institute of General Medical Sciences of the National Institutes of Health under award P20GM104357, and by the Mississippi INBRE, funded by an Institutional Development Award (IDeA) from the National Institutes of General Medical Sciences of the National Institutes of Health under grant number P20GM103476. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All protocols were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center. The care and handling of the animals were in accord with the National Institutes of Health guidelines for ethical animal treatment.
1. Lymphocyte Cell Isolation from Placentas
<ol>
	<li>Remove one placenta from the rat uterus (gestation day 19) and place in 10 mL of ice-cold PBS8.</li>
	<li>Place one placenta on a 100 &#181;m filter and sit in a Petri dish containing 13.5 mL of RPMI and 1.5 mL of &#160;FBS (total volume is 15 mL). Use the flat side of a syringe plunger to push the placenta through the filter into the Petri dish.</li>
	<li>Prepare three&#160;15 mL conical tubes for each tissue. Add 3 mL of density gradient medium (see Table of Materials) to each tube, then carefully overlay 5 mL of homogenized placenta into each tube.</li>
	<li>Centrifuge for 25 min at 300 x g at room temperature (RT) with no brake. Collect the thin white buffy layer with a transfer pipette. 
	NOTE: After centrifugation, 3 layers are visible, the red RPMI at the top, the white buffy layer in the middle, and the clear density gradient medium layer at the bottom. Use a transfer pipette to pull up the white buffy layer from the tube. Combine buffy layer from all tubes of the same placenta into 1 tube.</li>
	<li>Add 10 mL of RPMI to combined buffy layers. Centrifuge for 10 min, 300 x g, at 4 &#176;C and discard supernatant.</li>
</ol>
2. Isolation of Natural Killer Cells
<ol>
	<li>Resuspend cell pellet in 50 &#956;L of ice-cold PBS</li>
	<li>Add biotin-labeled CD3 antibody to pelleted cells according to the manufacturer&#8217;s protocol and mix well with a pipette. Place the tube in a tube rotator and incubate 20 min at 4 &#176;C.</li>
	<li>Add 1 mL of RPMI, centrifuge for 10 min at 400 x g and 4 &#176;C, and discard supernatant.</li>
	<li>Resuspend pellet in 1 mL of RPMI and combine with 150 &#956;L of magnetic beads in a 1.5 mL microcentrifuge tube. Place the microcentrifuge tube in a tube rotator and rotate while incubating for 30 min at 4 &#176;C. 
	NOTE: Pull out release buffer at this time and allow to reach RT in the biosafety cabinet.</li>
	<li>Place the tubes in the magnet for 1 min. Collect supernatant and save CD3- cell population in a 15 mL tube on ice. 
	NOTE: This is the CD3- population of cells.</li>
	<li>Remove the tube from the magnet and add 1 mL of RPMI. Mix cells and beads 5 times with a pipette.</li>
	<li>Repeat step 2.5.</li>
	<li>Centrifuge the CD3- population of cells for 10 min at 400 x g and 4 &#176;C and discard supernatant. Resuspend the cell pellet in 50 &#956;L of ice-cold PBS</li>
	<li>Add biotin-labeled CD161a antibody to CD3- cells according to the manufacturer&#8217;s protocol and mix well. Place tube in a tube rotator and incubate for 20 min at 4 &#176;C.</li>
	<li>Add 1 mL of RPMI, centrifuge for 10 min at 400 x g and 4 &#176;C, and discard supernatant. Resuspend pellet in 1 mL of RPMI and combine with 150 &#956;L of magnetic beads in a 1.5 mL microcentrifuge tube.</li>
	<li>Place microcentrifuge tube in a tube rotator and rotate while incubating for 30 min at 4 &#176;C.</li>
	<li>Place the tubes in a magnet for 1 min. Collect supernatant and discard CD3-/CD161a- cells.</li>
	<li>Remove tube from magnet and add 1 mL of RPMI. Mix cells and beads 5 times with a pipette.</li>
	<li>Repeat step 2.12.</li>
	<li>Remove microcentrifuge tubes from the magnet and add 1 mL of RT Release buffer. Place tube on tube rotator and rotate while incubating for 15 min at RT.</li>
	<li>Place tubes in magnet for 1 min. Collect supernatant in a new 15 mL conical tube on ice. 
	NOTE: This is the population of NK cells.</li>
	<li>Remove tube from magnet and add 1 mL of RT RPMI. Mix cells and beads 5 times with pipette.</li>
	<li>Repeat step 2.16, placing supernatant in the same tube. Mix well and take a 20 &#956;L sample to count cells 
	NOTE: Keep tube on ice.</li>
	<li>Centrifuge CD3-/CD161a+ cells for 10 min, 400 x g at 4 &#176;C, then remove supernatant. Resuspend the cells in RPMI (10% FBS, 1% Pen/Strep, 2 ng/ mL IL-2) and seed at a concentration of 3 x 105 cells/well in a 6-well plate in 2.5 mL of NK Cell Activation Media. Incubate cells for 48 h at 37 &#176;C, 5% CO2 in a humidified incubator.</li>
</ol>
3. Cytotoxicity Assay: Retrieving NK Cells or YAC1 from Culture or Passing Cells
NOTE: All steps must be conducted under the hood. All cell tubes must be kept on ice at all times.
<ol>
	<li>Use a glass serological pipet to collect YAC1 cells and media from flask, place in a 50 mL tube on ice, and mix well. Take 20 &#181;L to count cells.</li>
	<li>Spin the YAC1 cells for 10 min at 300 x g and 4 &#176;C. Count cells while these are spinning.</li>
	<li>Add trypsin/EDTA to each well in a NK cell 6-well plate. Tap the plate and place in incubator.</li>
	<li>After cells have incubated with trypsin/EDTA for ~5 min at 37 &#176;C, scrape the plate/flask with a sterile plate scraper. Add 1 mL of NK Cell Media to each well.</li>
	<li>Collect cells and media with serological pipet and collect in a 15 mL centrifuge tube. Take 20 &#181;L to count cells.</li>
	<li>Spin the NK cells for 10 min, 400 x g at 4 &#176;C. Count the sample of cells from step 3.5 during this centrifugation. 
	NOTE: View the culture plates under a microscope before discarding them to make sure there are no more cells adhered to the bottom of the wells. Since the experiment uses NK cells and YAC1 cells, be sure to count each one separately.</li>
	<li>Count cells and resuspend at the concentrations determined in optimization trials to test for the appropriate number of target:effector ratio. This will be achieved by re-suspending the pellet in their corresponding media to make the following cell concentrations: YAC1 at 4 x 105 cells/mL and NK cells at 2 x 107 cells/mL.</li>
</ol>
4. Cytotoxicity Assay: Assay Protocol
<ol>
	<li>Use a round bottom, culture treated 96-well plate to set up the plate as suggested in Table 1. This table shows the experimental controls and 3 sets of NK experimental columns. This can be expanded to a total of 10 NK experimental columns in a 96-well plate.</li>
	<li>Centrifuge the assay plate at 250 x g for 4 min to be certain that the effector and target cells are in contact. Incubate the 96-well plate for 5 hours in a humidified chamber incubator at 37 &#176;C, 5% CO2 to achieve ample contact between target and effector cells and target cell lysis by effector cells. 
	NOTE: The protocol can be paused here.</li>
	<li>45 min prior to harvesting supernatants, add 10 &#181;L of 10x Lysis Solution to the Target Cell Maximum LDH Release wells (Wells 1E, 1F, 1G, and 1H) and place the plate back in the humidified chamber.</li>
	<li>After the incubation time is completed, centrifuge the plate at 250 x g for 4 min. Using a multichannel pipettor, transfer 50 &#181;L aliquots from all wells to a fresh 96-well flat-bottom assay plate. 
	NOTE: Do not touch the bottom of the wells so that cells are not transferred into the fresh assay plate. Do not use a cultured treated plate as the fresh assay plate.</li>
	<li>Make Assay Reagent.
	<ol>
		<li>After Assay Buffer has reached room temperature, add 12 mL of Assay Buffer to one Substrate Mix bottle. Invert and shake gently until completely dissolved. 1 bottle is enough for two 96-well plates. 
		NOTE: Assay Buffer should be protected from light while being thawed. Mix immediately prior to use.</li>
	</ol>
	</li>
	<li>Add 50 &#181;L of Assay Reagent to all wells in the assay plate from step 4.6. Cover the plate with foil to protect it from light and incubate for 30 min at room temperature. 
	NOTE: Newly made Assay Reagent should be stored in a freezer. Reagent is good for approximately 8 weeks.</li>
	<li>Add 50 &#181;L of Stop Solution to each well. Read plate within 1 hour after adding Stop Solution and record the absorbance at 490 nm. Representative data is shown in Table 2.</li>
</ol>
5. Calculation of Results
<ol>
	<li>Calculate the average absorbance value from the Culture Medium Background wells and subtract from all absorbance values for Experimental, Target Cell Spontaneous LDH Release and Effector Cell Spontaneous LDH Release wells.</li>
	<li>Calculate the average absorbance values for the Volume Correction Control wells and subtract from the absorbance values acquired for the Target Cell Maximum LDH Release Control wells.</li>
	<li>Use the corrected values from Steps 5.1 and 5.2&#160;in the following formula to calculate percent cytotoxicity for each effector:target well. 
	<img alt="Equation 1" src="/files/ftp_upload/58961/58961eq1.jpg" /></li>
</ol>

<ol>
	<li>Cornelius, D. C., Cottrell, J., Amaral, L. M., LaMarca, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+Mediators:+A+causal+link+to+hypertension+during+pregnancy-+Studies+in+Preeclampsia.">Inflammatory Mediators: A causal link to hypertension during pregnancy- Studies in Preeclampsia.</a> British Journal of Pharmacology. , (2018).</li><li>Elfarra, 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=Natural+killer+cells+mediate+pathophysiology+in+response+to+reduced+uterine+perfusion+pressure.">Natural killer cells mediate pathophysiology in response to reduced uterine perfusion pressure.</a> Clinical Science. 131 (23), 2753-2762 (2017).</li><li>Brunner, K. T., Mauel, J., Cerottini, J. C., Chapuis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assay+of+the+lytic+action+of+immune+lymphoid+cells+on+51-Cr-labelled+allogeneic+target+cells+in+vitro;+inhibition+by+isoantibody+and+by+drugs.">Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs.</a> Immunology. 14 (2), 181-196 (1968).</li><li>Kim, G. G., Donnenberg, V. S., Donnenberg, A. D., Gooding, W., Whiteside, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multiparametric+flow+cytometry-based+cytotoxicity+assay+simultaneously+immunophenotypes+effector+cells:+comparisons+to+a+4+h+51Cr-release+assay.">A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.</a> Journal of Immunological Methods. 325 (1-2), 51-66 (2007).</li><li>Somanchi, S. S., McCulley, K. J., Somanchi, A., Chan, L. L., Lee, 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=A+Novel+Method+for+Assessment+of+Natural+Killer+Cell+Cytotoxicity+Using+Image+Cytometry.">A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.</a> PLoS One. 10 (10), 0141074 (2015).</li><li>Neri, S., Mariani, E., Meneghetti, A., Cattini, L., Facchini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcein-acetyoxymethyl+cytotoxicity+assay:+standardization+of+a+method+allowing+additional+analyses+on+recovered+effector+cells+and+supernatants.">Calcein-acetyoxymethyl cytotoxicity assay: standardization of a method allowing additional analyses on recovered effector cells and supernatants.</a> Clinical and Diagnostic Laboratory Immunology. 8 (6), 1131-1135 (2001).</li><li>Karimi, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+cytotoxicity+by+bioluminescence+imaging+outperforms+the+standard+chromium-51+release+assay.">Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay.</a> PLoS One. 9 (2), 89357 (2014).</li><li>Caporossi, C., Nogueira, P. L., Marques, J. C., Assis, R. M., Aguilar-Nascimento, J. E. <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+the+gastroschisis+experimental+model+and+the+influence+of+the+mother's+diet+enriched+with+glutamine+in+the+fetal+morphology.">Validation of the gastroschisis experimental model and the influence of the mother's diet enriched with glutamine in the fetal morphology.</a> Acta Cir&#250;rgica Brasileira. 29 (3), 158-165 (2014).</li><li>Lv, L. 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=Functional+distinction+of+rat+liver+natural+killer+cells+from+spleen+natural+killer+cells+under+normal+and+acidic+conditions+in+vitro.">Functional distinction of rat liver natural killer cells from spleen natural killer cells under normal and acidic conditions in vitro.</a> Hepatobiliary and Pancreatic Diseases International. 11 (3), 285-293 (2012).</li><li>Flieger, D., 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+novel+non-radioactive+cellular+cytotoxicity+test+based+on+the+differential+assessment+of+living+and+killed+target+and+effector+cells.">A novel non-radioactive cellular cytotoxicity test based on the differential assessment of living and killed target and effector cells.</a> Journal of Immunological Methods. 180 (1), 1-13 (1995).</li><li>Jang, Y. 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No conflicts of interest, financial or otherwise, are declared by the authors.

There are a number of important key notes to consider for optimal results. The sterility of the cells utilized is very important. After collection of the placenta, it is important that preparation and isolation of the NK cells are performed under sterile conditions in a biosafety cabinet. Furthermore, because all cells release LDH upon cellular damage, care should be taken to obtain a high viability of NK cells after isolation and during the co-culture process. Too much spontaneous LDH release from the NK cells can often...

Preeclampsia is a hypertensive disorder of pregnancy characterized by fetal growth restriction, end organ damage and chronic immune activation. Chronic immune activation in women with preeclampsia leads to increased circulating and placental inflammatory cytokines, an imbalance in CD4+ T Cells populations, and an increased population of activated Natural Killer (NK) cells1. Studies recently published by our lab demonstrate a role for NK cells in causing some of the pathophysiology associated with preeclampsia in the Reduced Uterine Perfusion Pressure (RUPP) rat model of preeclampsia. Using flow cytometry to measure surface expression of activation markers on NK cells, an increased population of activated NK cells in the circulation and placentas of RUPP rats compared to normal pregnant (NP) rats was observed2.
To confirm the flow cytometry observations, functional studies to assess the cytotoxic activity of NK cells isolated from the placentas of NP and RUPP rats were performed. There are several methods available for the assessment of cytotoxic function of cytotoxic CD8+ T cells and NK cells. The gold standard for functional cytotoxic analysis is the chromium release assay3. Other developed protocols utilized include flow cytometry4, image cytometry5, calcein release6, and most recently bioluminescence7. This video will provide a detailed protocol on using the well-established lactate dehydrogenase (LDH) release assay to measure cytotoxic function of NK cells using a commercially available LDH cytotoxicity assay kit.

<table border="0" cellpadding="0" cellspacing="0" style="width:751px;" width="751">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">1.5 mL Eppendorf tube</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">5408129</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 &#181;L Filter</td>
			<td>Fisher</td>
			<td style="width:119px;">22363549</td>
			<td>Nylon Mesh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL conical tube</td>
			<td>Fisher</td>
			<td>0553859A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 mL syringe</td>
			<td>Fisher</td>
			<td>14823436</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical tube</td>
			<td>Fisher</td>
			<td>7203510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well cell culture plate</td>
			<td>Corning</td>
			<td>720083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well Tissue Culture Plate</td>
			<td>CELLTREAT</td>
			<td>229190</td>
			<td>Sterile, Round Bottom</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AOPI</td>
			<td>Nexcelom</td>
			<td style="width:119px;">CS201065ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell scraper</td>
			<td>Fisher</td>
			<td>8100241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellometer Disposable Counting Chambers</td>
			<td style="width:123px;">Nexcelom</td>
			<td style="width:119px;">CHT4-SD100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cellometer Vision Image Cytometer</td>
			<td style="width:123px;">Nexcelom</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytotox 96 Non-Radioactive Cytotoxicity Assay Kit</td>
			<td>Promega</td>
			<td>G1780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dynabeads Flowcomp Flexi Kit</td>
			<td>Invitrogen</td>
			<td>11061D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-2 Magnet</td>
			<td>Invitrogen</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td>EDS-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Atlanta Biologicals</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Cytometry Tube</td>
			<td>Corning</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lymphoprep</td>
			<td>Fisher</td>
			<td>NC0460539</td>
			<td>Density gradient medium; 4 x 250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Fisher</td>
			<td>SH3025801</td>
			<td>10 x 500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes</td>
			<td>Fisher</td>
			<td>9720500</td>
			<td>Without Pad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified Mouse anti-Rat CD161a</td>
			<td>BD Biosciences</td>
			<td>555006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified Mouse anti-Rat CD3</td>
			<td>BD Biosciences</td>
			<td>554829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Rat IL-2</td>
			<td>R&#38;D Systems</td>
			<td>502-RL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>Gibco</td>
			<td>11875135</td>
			<td>1640 Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T25 flask</td>
			<td>Corning</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td>ThermoFisher</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">YAC-1 cell</td>
			<td>ATCC</td>
			<td>TIB-160</td>
			<td></td>
		</tr>
	</tbody>
</table>

a plate-based cytotoxicity assay for the assessment of rat placental natural killer cell cytolytic function

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have demonstrated an altered population of circulating and decidual NK cells in women who suffer from adverse pregnancy complications such as recurrent miscarriage and preeclampsia. Studies from our group have shown that hypertension in pregnancy is associated with an increased population of activated NK cells in the placenta based on the expression of surface activation markers. This manuscript provides a detailed protocol to assess the cytotoxic function of NK cells isolated from placentas in a preeclampsia-like animal model of surgically induced placental ischemia. The following steps are described in detail: generation of single cell suspension, NK cell isolation, ex vivo stimulation, effector:target cell co-culture, and the cytotoxicity assay.

Placental NK cells obtained from NP and RUPP rats were incubated for 5 h with target cells in their respective medias at a ratio of 50:1 (NK:target). Absorbance was recorded at 490 nm and the raw data is shown in Table 2. The average absorbance of the Culture Medium Background and the Volume Correction control wells were calculated. These averages were subtracted from the appropriate wells indicated in the manufacturer&#8217;s protocol and are represented in Table 3. The corrected values...

Watch this Scientific Journal Video about A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function at JoVE.com

A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function

Here, we provide a detailed methodology to isolate and assess cytotoxic function of natural killer cells from placentas by a colorimetric plate assay. The reduced uterine perfusion pressure rat model of placental ischemia was chosen to demonstrate the antibody-mediated isolation and assessment of the cytotoxic function of natural killer cells.

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have ...

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Immunology and Infection

5.7K Views.  University of Mississippi Medical Center. Here, we provide a detailed methodology to isolate and assess cytotoxic function of natural killer cells from placentas by a colorimetric plate assay. The reduced uterine perfusion pressure rat model of placental ischemia was chosen to demonstrate the antibody-mediated isolation and assessment of the cytotoxic function of natural killer cells.

Video: A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

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

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

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

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

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

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...