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. Y., 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=An+improved+flow+cytometry-based+natural+killer+cytotoxicity+assay+involving+calcein+AM+staining+of+effector+cells.">An improved flow cytometry-based natural killer cytotoxicity assay involving calcein AM staining of effector cells.</a> Annals of Clinical Laboratory Science. 42 (1), 42-49 (2012).</li></ol>

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

a-plate-based-cytotoxicity-assay-for-assessment-rat-placental-natural

Research

JoVE Journal

Immunology and Infection

5.8K Views.  University of Mississippi Medical Center. Deze methode helpt bij het vergelijken van de cytotoxische activiteit van verschillende celtypen met behulp van een eenvoudige, goedkope methode die geen gespecialiseerde apparatuur vereist. Het belangrijkste voordeel van deze techniek is dat het niet het gebruik van gevaarlijke stoffen vereist, het is goedkoop en vereist niet het gebruik van gespecialiseerde apparatuur. Deze methode kan worden toegepast om cytotoxische activiteit van andere celtypen zoals CD8 plus cytolytische cellen te mete...

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...

Developmental Biology

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

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, ...

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

Here we design two complementary mass cytometry (CyTOF) panels and optimize a CyTOF staining protocol with the aim of profiling the natural killer cell receptor and ligand repertoire in the setting of viral infections.

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

Medicine

Calcein Acetoxymethyl Staining to Evaluate Natural Killer Cell-Mediated Cytotoxicity toward Cancer Cells

Source: Chava, S. et al., <a href="https://app.jove.com/v/60714">Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells.</a> J. Vis. Exp. (2020)
This video demonstrates an assay to measure the cytotoxicity of natural killer cells against cancer cells using the Calcein AM stain. The reduced number of fluorescent cancer cells following co-culture with natural killer cells indicates natural killer cell-mediated cytotoxicity toward cancer cells.

Source: Chava, S. et al., Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

Natural Killer Cell-Mediated Cytotoxicity Assay Sample Preparation for Flow Cytometric Analysis

Source: McBride, J. E., et al. <a href="https://app.jove.com/v/54779">Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood.</a> J. Vis. Exp. (2017).
This video demonstrates the lytic activity of natural killer cells against fluorescently-stained target cells. The method enables NK cell-induced cytotoxicity measurement by staining dead cells and using a flow cytometer to quantify live and dead target cells.

1.&#160;Preparation of Controls

	Transfer the following into separate and appropriately labeled 1.5 ml tubes:
	
		Add 500 &#181;L of fresh IMDM ...

Cellular Immunodiagnostics

An In Vitro Assay for Evaluating Natural Killer Cell Cytotoxicity against Cancer Cells

Source: Baik, C. H., et al. <a href="https://app.jove.com/v/58961">A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function.</a> J. Vis. Exp. (2019)
This video demonstrates performing a lactate dehydrogenase release assay to determine the cytotoxicity of natural killer (NK) cells against the target cancer cells. The assay quantifies the release of the cytoplasmic lactate dehydrogenase enzyme from cancer cells by measuring the absorbance of the red-colored product formed.

1. Cytotoxicity Assay: Assay Protocol

	Use a round bottom, culture treated 96-well plate to set up the plate as suggested in Table 1. This table shows ...

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

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