We would like to thank the VIB Bioimaging Core&#160;for training, support, and access to the instrument park. J.N. is supported by a PhD fellowship from the Research Foundation Flanders (FWO). Research in the J.M. group was supported by an Odysseus II Grant (G0H8618N), EOS INFLADIS (40007512), a junior research grant (G031022N) from the Research Foundation Flanders (FWO), a CRIG young investigator proof-of-concept grant, and by Ghent University. Research in the P.V. group was supported by EOS MODEL-IDI (30826052), EOS INFLADIS (40007512), FWO senior research grants (G.0C76.18N, G.0B71.18N, G.0B96.20N, G.0A9322N), Methusalem (BOF16/MET_V/007), iBOF20/IBF/039 ATLANTIS, Foundation against Cancer (F/2016/865, F/2020/1505), CRIG and GIGG consortia, and VIB.

1. Preparation of biotinylated tyramide
<ol>
	<li>Prepare biotinylated tyramide starting from biotin-tyramide. To make a 10 mM stock solution, dissolve 3.6 mg of biotin-tyramide in 1 mL of DMSO. Store the dissolved product in aliquots at &#8722;20 &#176;C to preserve the quality.</li>
</ol>
2. Maintaining HT-29 cells in culture
NOTE: ZBP1-expressing HT-29 were generated by transduction with a lentivector27 encoding human ZBP1.
<ol>
	<li>Keep ZBP1-expressing HT-29 cells in McCoy&#39;s 5A medium supplemented with L-glutamine, sodium-pyruvate, and 10% fetal bovine serum (from now on referred to as full medium) and maintain in an incubator at 37 &#176;C with 5% carbon dioxide. Ideally, use cells of a low passage number (less than 10). Leave the cells to recover for at least 5 days after the thaw cycle before the start of the experiment.</li>
	<li>To detach the cells from the culture flask, remove the medium and wash the cells with 5 mL of PBS (preheated to 37 &#176;C). Next, add the appropriate amount of trypsin/EDTA (0.05% trypsin and 0.032% EDTA, respectively, preheated to 37 &#176;C) to the cells (2 mL for a T75 flask, 3 mL for a T175 flask).</li>
	<li>Incubate the cells with trypsin/EDTA for up to 10 min in an incubator at 37 &#176;C with 5% carbon dioxide. Afterward, tap the flask and visually check if the cells detached from the flask using a microscope with 4x-20x objective magnification.</li>
	<li>If the cells are not fully detached, incubate for another 5 min at 37 &#176;C. When the cells are detached, stop the enzymatic reaction by adding 6 mL of full medium to the flask.</li>
	<li>Gather the cell suspension in a 15 mL tube. Count the cells using&#160;a trypan blue stain to assess viability; a 1:5 dilution is recommended. Only proceed with the experiment if the viability of the cells exceeds 90%.</li>
</ol>
3. Starting the experiment, seeding, and stimulation of the cells
<ol>
	<li>Seed 90,000 ZBP1-expressing HT-29 cells in full medium in a 1 cm&#178; surface area well plate that allows high end microscopy. Use an end volume of 200 &#181;L per well.</li>
	<li>Incubate the cells overnight at 37 &#176;C with 5% carbon dioxide. Take into consideration that some extra wells need to be seeded for staining controls, which is explained in more detail in step 5.6.</li>
	<li>When the cells reach 70%-80% confluency, add a necroptosis-inducing stimulus to the cells. Here, the cells were infected with HSV-1 ICP6mutRHIM (multiplicity of infection [MOI] of 5, defined as plaque forming units (pfu) divided by the number of cells; the pfu was quantified using a plaque assay on Vero cells) for 6 h, 8 h, or 10 h.</li>
	<li>As a positive control for necroptosis induction, stimulate the cells for 4 h with a necroptosis-inducing cocktail containing 30 ng/mL TNF, 20 &#181;M of the pan-caspase inhibitor zVAD-fmk, and 5 &#181;M of the SMAC mimetic BV6.</li>
	<li>As a negative control for p-RIPK3 (S227) staining, include GSK&#39;840 (1 &#181;M) to inhibit RIPK3 kinase activity and prevent the autophosphorylation of S227. Ideally, add the inhibitor in an untreated condition and after necroptosis stimulation. The inhibitor can be added simultaneously with viral infection.</li>
	<li>Prepare the stimulations in full medium, preheated to 37 &#176;C. Use an end volume of 200 &#181;L per well for all stimulations.</li>
</ol>
4. Fixing the cells
<ol>
	<li>Remove the medium and wash the cells with 200 &#181;L of 1x PBS. Then, add 150 &#181;L of 4% PFA (already equilibrated at room temperature) to the cells and incubate at room temperature for 30 min. 
	NOTE: If you are using cells that detach easily, the fixation of the cells can be optimized as follows. Remove 100 &#181;L of medium from the well, so a volume of 100 &#181;L remains on the plate. Add 100 &#181;L of 4% PFA to the cells. Incubate the cells for 5 min at room temperature. Afterward, remove the medium/fixative from the wells and replace with 150 &#181;L of 4% PFA. Incubate the cells for another 20 min at room temperature to ensure complete fixation of the cells.</li>
	<li>After fixation, remove the 4% PFA and wash the cells 3x with 200 &#181;L of 1x PBS. The samples can be stored in an excess (&#62;200 &#181;L) of 1x PBS at 4 &#176;C overnight until further processing.</li>
</ol>
5. Permeabilization and primary staining
<ol>
	<li>Remove the PBS and add 100 &#181;L of permeabilization buffer (0.5% Triton X-100 in PBS). Incubate at room temperature for 30 min.</li>
	<li>Remove the permeabilization buffer and, subsequently, wash the wells with 100 &#181;L of wash buffer (0.1% Triton X-100 in PBS). Incubate the imaging chamber with wash buffer for 5 min at room temperature on a tilting laboratory shaker (20-30 rocking motions per min).</li>
	<li>To prevent non-specific binding of primary antibodies, add 100 &#181;L of blocking medium and incubate at room temperature for 2 h. Alternatively, this blocking step may be replaced by blocking with 3% BSA, 0.1% Triton-X-100 in PBS for 1 h at room temperature.</li>
	<li>Wash the wells 3x with 100 &#181;L of wash buffer (0.1% Triton X-100 in PBS). Incubate the wash steps for 5 min at room temperature on a tilting laboratory shaker.</li>
	<li>After removing the wash buffer, add the primary antibodies to the imaging chamber and incubate overnight at 4 &#176;C. Use an end volume of 100 &#181;L to cover the well. During overnight incubation at 4&#176;C, do not place the imaging chamber on a tilting laboratory shaker. 
	NOTE: Anti p-MLKL (S358; dilution: 1:200) and anti-p-RIPK3 (S227; dilution: 1:200) share rabbit as a host species and should, therefore, not be combined in one well. To monitor the viral infection, combine a primary antibody against a viral protein of choice with p-MLKL (S358) or p-RIPK3 (S227). Here, the cells were infected with HSV-1 infection followed by ICP0 staining (dilution: 1:50, host species: mouse).</li>
	<li>Take into consideration the necessary staining controls at this point. Always include a no primary antibody condition to visualize the potential background of the TSA amplification step to set the masking threshold (see step 9). 
	&#8203;NOTE: In case of a co-staining protocol (e.g., combining p-MLKL [S358] or p-RIPK3 [S227] with an antibody against a viral protein) use single stains as well. The use of single stains is important to correct for potential bleed-through signal in other imaging channels.</li>
</ol>
6. Tyramide signal amplification (TSA)
<ol>
	<li>Remove the primary antibody mix and wash wells 3x with 100 &#181;L of wash buffer (0.1% Triton X-100 in PBS). Incubate the wash steps for 5 min at room temperature on a tilting laboratory shaker.</li>
	<li>Remove the wash buffer and add 100 &#181;L of HRP-labeled secondary antibody recognizing the species of primary antibody that needs to be amplified. To amplify the p-RIPK3 (S227) or p-MLKL (S358) signal, add 100 &#181;L of anti-rabbit-HRP and incubate for 30 min at room temperature on a tilting laboratory shaker. 
	NOTE: Only p-MLKL (S358) or p-RIPK3 (S227) will be amplified. The staining of a viral protein will be visualized using a standard indirect staining method.</li>
	<li>Afterward, wash the wells 3x with 100 &#181;L of wash buffer (0.1% Triton X-100 in PBS). Incubate the wash steps for 5 min at room temperature on a tilting laboratory shaker.</li>
	<li>In the next steps, add the biotinylated tyramide to the microscopic plate. Using the HRP-group coupled to the secondary antibodies, the enzymatic reaction will trigger the formation of tyramide radicals in close proximity to the primary target (here, p-MLKL [S358] or p-RIPK3 [S227]).</li>
	<li>To activate the enzymatic activity of HRP, add an oxidizing substrate together with the biotinylated tyramide. To achieve this, supplement the TSA buffer (0.1 M boric acid [pH 8.5]) with 0.03 M H2O2; specifically, take 5 mL of TSA buffer and add 5 &#181;L of 30% H2O2.</li>
	<li>Now, dilute the biotin-tyramide in H2O2-supplemented TSA buffer ranging from 1:1,000 to 1:20,000. 
	NOTE: The dilution factor of the biotin-tyramide needs to be optimized per batch.</li>
	<li>Remove the wash buffer from the wells and add diluted biotin-tyramide to the wells to an end volume of 100 &#181;L. Incubate at room temperature for 10 min on a tilting laboratory shaker.</li>
	<li>Next, wash the wells 3x with 100 &#181;L of wash buffer (0.1% Triton X-100 in PBS). Incubate the wash steps for 5 min at room temperature on a tilting laboratory shaker.</li>
</ol>
7. Fluorophores
NOTE: Since the signal of the primary antibody is converted to a biotin-group, p-MLKL (S358) and p-RIPK3 (S227) are visualized using streptavidin coupled to a fluorophore (fluorophore 568, dilution: 1:500). Additionally, the nuclei are stained with DAPI (5 &#181;g/mL). If a viral protein is included in the staining protocol, include a suitable fluorescently labeled secondary antibody against the host species of your primary antibody. In the representative results, a mouse anti-ICP0 was used. As a secondary antibody goat anti-mouse coupled to fluorophore 633 (dilution: 1:1,000) was included.
<ol>
	<li>Make the staining mix in wash buffer (0.1% Triton X-100 in PBS) containing the antibodies and stains mentioned above. Remove the wash buffer, add 100 &#181;L of staining mix, and incubate at room temperature for 1 h on a tilting laboratory shaker. Keep the imaging chamber shielded from light from this step onward.</li>
	<li>Next, wash the wells 2x with wash buffer (0.1% Triton X-100 in PBS). Incubate the wash steps for 5 min at room temperature on a tilting laboratory shaker.</li>
	<li>Finally, rinse the wells 2x with 1x PBS. Store the samples in an excess (&#62; 200 &#181;L) of 1x PBS until imaging. Alternatively, submerge the samples in mounting medium to preserve the staining. The imaging chamber is now ready to be visualized on a confocal microscope.</li>
</ol>
8. Imaging using a confocal microscope
<ol>
	<li>Turn on the confocal microscope and lasers at least 10 min before imaging.</li>
	<li>Use an immersion objective for sensitivity. Preferably, use 40x or 63x objective magnification. Before imaging, clean the immersion objectives with lens cleaner using appropriate cotton balls. Dirty objectives (e.g., due to dust) can result in lower quality images.</li>
	<li>Put an excess amount of oil on the bottom of the imaging chamber. Additionally, add a drop of oil on the objective of choice.</li>
	<li>Put the imaging chamber on the microscope. Find the focus field using DAPI staining or using brightfield imaging. If necessary, move around the imaging chamber to ensure proper spread of the immersion oil.</li>
	<li>Set up the necessary imaging tracks on the microscope. Depending on the microscope, the following steps may vary (Table 1). 
	NOTE: When setting up the imaging tracks, program the tracks so that the nuclear staining is measured last. The 405 nm laser may contribute to photobleaching and, thus, the loss of specific signal in all the channels.</li>
	<li>To analyze a complete cell for the presence of p-RIPK3 (S227) or p-MLKL (S358), measure z-stacks that span the height of the cell. Here, the z-stacks were made of 40 slices every 0.16 &#181;m, resulting in a range of 6.22 &#181;m.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Track</td>
			<td>Laser</td>
			<td>Beam splitter</td>
			<td>Filter</td>
		</tr>
		<tr>
			<td rowspan="2">pRIPK3 (S227) /pMLKL (S358)</td>
			<td rowspan="2">561</td>
			<td>MBS -405 +</td>
			<td rowspan="2">BP 570-620 + LP645</td>
		</tr>
		<tr>
			<td>MBS 488/561/633 + SBS SP615</td>
		</tr>
		<tr>
			<td rowspan="2">Viral Gene: ICP0</td>
			<td rowspan="2">633</td>
			<td>MBS -405 +</td>
			<td rowspan="2">BP 420-480 + LP605</td>
		</tr>
		<tr>
			<td>MBS 488/561/633 + SBS LP570</td>
		</tr>
		<tr>
			<td rowspan="2">Nucleus: DAPI</td>
			<td rowspan="2">405</td>
			<td>MBS -405 +</td>
			<td rowspan="2">BP 420-480 + BP 495-550</td>
		</tr>
		<tr>
			<td>MBS 488/561/633</td>
		</tr>
	</tbody>
</table>
Table 1: Imaging tracks for cell visualization.
9. Data analysis and quantification
<ol>
	<li>Upload the microscopic images to the software. Use the extended focus to visualize all the information of the z-stack in a 2D image.</li>
	<li>Next, program the analysis protocol in order to extract the following information: the number of cells per image, the sum of voxels showing p-RIPK3 (S227)+ or p-MLKL (S358)+ staining. A voxel represents a three-dimensional volume, defined as the three-dimensional equivalent of a pixel.</li>
	<li>To quantify the number of cells per image, segment the nucleus. To reduce noise in the segmentation results, add a size limitation to the segmentation (&#62;100 &#181;m&#179;). The number of segmented nuclei represents the number of cells in the image.</li>
	<li>To quantify the amount of p-RIPK3 (S227)+ or p-MLKL (S358)+ voxels, program a threshold-based segmentation. Set the threshold so that no signal is picked up in the no primary antibody (NP) images.</li>
	<li>Further adapt the threshold using the images from the untreated mock condition. To ensure sensitive detection of the signal increase due to necroptosis stimulation, limit the pick-up of p-RIPK3 (S227)+ or p-MLKL (S358)+ voxels in mock conditions by setting a higher threshold. Always check the programmed analysis protocol in several images from the mock condition and necroptosis-inducing viral infection.</li>
	<li>Run the analysis on all image sets. Export the voxel quantification and nucleus segmentation in a .txt file for further processing in spreadsheet software.</li>
	<li>Make a pivot table of the data showing the image name, the nucleus quantification, and the sum of detected voxels.</li>
	<li>Next, divide the sum of positive voxels by the count of cells in the image. This results in a relative value of positive voxels per cell. To visualize the fold increase, divide the relative value of p-RIPK3 (S227)+ or p-MLKL (S358)+ voxels per cell by the median of the untreated condition (mock).</li>
</ol>

<ol>
	<li>Meng, Y., Sandow, J. J., Czabotar, P. E., Murphy, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+necroptosis+by+post-translational+modifications.">The regulation of necroptosis by post-translational modifications.</a> Cell Death &amp; Differentiation. 28 (3), 861-883 (2021).</li><li>Petrie, E. J., Czabotar, P. E., Murphy, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+basis+of+necroptotic+cell+death+signaling.">The structural basis of necroptotic cell death signaling.</a> Trends in Biochemical Sciences. 44 (1), 53-63 (2019).</li><li>Mocarski, E. S., Guo, H., Kaiser, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis:+The+Trojan+horse+in+cell+autonomous+antiviral+host+defense.">Necroptosis: The Trojan horse in cell autonomous antiviral host defense.</a> Virology. 479-480, 160-166 (2015).</li><li>Nailwal, H., Chan, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis+in+anti-viral+inflammation.">Necroptosis in anti-viral inflammation.</a> Cell Death &amp; Differentiation. 26 (1), 4-13 (2019).</li><li>Meng, 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=Human+RIPK3+maintains+MLKL+in+an+inactive+conformation+prior+to+cell+death+by+necroptosis.">Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis.</a> Nature Communications. 12 (1), 6783 (2021).</li><li>Samson, A. L., Garnish, S. E., Hildebrand, J. M., Murphy, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location,+location,+location:+A+compartmentalized+view+of+TNF-induced+necroptotic+signaling.">Location, location, location: A compartmentalized view of TNF-induced necroptotic signaling.</a> Science Signalling. 14 (668), (2021).</li><li>Koehler, 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=Vaccinia+virus+E3+prevents+sensing+of+Z-RNA+to+block+ZBP1-dependent+necroptosis.">Vaccinia virus E3 prevents sensing of Z-RNA to block ZBP1-dependent necroptosis.</a> Cell Host Microbe. 29 (8), 1266-1276 (2021).</li><li>Balachandran, S., Mocarski, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+Z-RNA+triggers+ZBP1-dependent+cell+death.">Viral Z-RNA triggers ZBP1-dependent cell death.</a> Current Opinion in Virology. 51, 134-140 (2021).</li><li>Kuriakose, T., Kanneganti, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZBP1:+Innate+sensor+regulating+cell+death+and+inflammation.">ZBP1: Innate sensor regulating cell death and inflammation.</a> Trends in Immunology. 39 (2), 123-134 (2018).</li><li>Maelfait, J., Liverpool, L., Rehwinkel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+sensors+and+programmed+cell+death.">Nucleic acid sensors and programmed cell death.</a> Journal of Molecular Biology. 432 (2), 552-568 (2020).</li><li>Verdonck, S., Nemegeer, J., Vandenabeele, P., Maelfait, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+manipulation+of+host+cell+necroptosis+and+pyroptosis.">Viral manipulation of host cell necroptosis and pyroptosis.</a> Trends in Microbiology. 30 (6), 593-605 (2022).</li><li>Guo, 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=Herpes+simplex+virus+suppresses+necroptosis+in+human+cells.">Herpes simplex virus suppresses necroptosis in human cells.</a> Cell Host &amp; Microbe. 17 (2), 243-251 (2015).</li><li>Yu, X., 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=Herpes+simplex+virus+1+(HSV-1)+and+HSV-2+mediate+species-specific+modulations+of+programmed+necrosis+through+the+viral+ribonucleotide+reductase+large+subunit+R1.">Herpes simplex virus 1 (HSV-1) and HSV-2 mediate species-specific modulations of programmed necrosis through the viral ribonucleotide reductase large subunit R1.</a> Journal of Virology. 90 (2), 1088-1095 (2016).</li><li>Huang, Z., 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=RIP1/RIP3+binding+to+HSV-1+ICP6+initiates+necroptosis+to+restrict+virus+propagation+in+mice.">RIP1/RIP3 binding to HSV-1 ICP6 initiates necroptosis to restrict virus propagation in mice.</a> Cell Host &amp; Microbe. 17 (2), 229-242 (2015).</li><li>Guo, 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=Species-independent+contribution+of+ZBP1/DAI/DLM-1-triggered+necroptosis+in+host+defense+against+HSV1.">Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1.</a> Cell Death &amp; Disease. 9 (8), 816 (2018).</li><li>Devos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+of+endogenous+nucleic+acids+by+ZBP1+induces+keratinocyte+necroptosis+and+skin+inflammation.">Sensing of endogenous nucleic acids by ZBP1 induces keratinocyte necroptosis and skin inflammation.</a> The Journal of Experimental Medicine. 217 (7), 20191913 (2020).</li><li>Jiao, 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=Z-nucleic-acid+sensing+triggers+ZBP1-dependent+necroptosis+and+inflammation.">Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation.</a> Nature. 580 (7803), 391-395 (2020).</li><li>de Reuver, R., 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=ADAR1+prevents+autoinflammation+by+suppressing+spontaneous+ZBP1+activation.">ADAR1 prevents autoinflammation by suppressing spontaneous ZBP1 activation.</a> Nature. 607 (7920), 784-789 (2022).</li><li>Jiao, 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=ADAR1+averts+fatal+type+I+interferon+induction+by+ZBP1.">ADAR1 averts fatal type I interferon induction by ZBP1.</a> Nature. 607 (7920), 776-783 (2022).</li><li>Hubbard, N. W., 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=ADAR1+mutation+causes+ZBP1-dependent+immunopathology.">ADAR1 mutation causes ZBP1-dependent immunopathology.</a> Nature. 607 (7920), 769-775 (2022).</li><li>Zhang, T., 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=ADAR1+masks+the+cancer+immunotherapeutic+promise+of+ZBP1-driven+necroptosis.">ADAR1 masks the cancer immunotherapeutic promise of ZBP1-driven necroptosis.</a> Nature. 606 (7914), 594-602 (2022).</li><li>Adams, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotin+amplification+of+biotin+and+horseradish+peroxidase+signals+in+histochemical+stains.">Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains.</a> The Journal of Histochemistry and Cytochemistry. 40 (10), 1457-1463 (1992).</li><li>Speel, E. J., Hopman, A. H., Komminoth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyramide+signal+amplification+for+DNA+and+mRNA+in+situ+hybridization.">Tyramide signal amplification for DNA and mRNA in situ hybridization.</a> Methods in Molecular Biology. 326, 33-60 (2006).</li><li>Clutter, M. R., Heffner, G. C., Krutzik, P. O., Sachen, K. L., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyramide+signal+amplification+for+analysis+of+kinase+activity+by+intracellular+flow+cytometry.">Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry.</a> Cytometry A. 77 (11), 1020-1031 (2010).</li><li>Dopie, J., Sweredoski, M. J., Moradian, A., Belmont, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyramide+signal+amplification+mass+spectrometry+(TSA-MS)+ratio+identifies+nuclear+speckle+proteins.">Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins.</a> The Journal of Cell Biology. 219 (9), 201910207 (2020).</li><li>Samson, A. L., 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+toolbox+for+imaging+RIPK1,+RIPK3,+and+MLKL+in+mouse+and+human+cells.">A toolbox for imaging RIPK1, RIPK3, and MLKL in mouse and human cells.</a> Cell Death and Differentiation. 28 (7), 2126-2144 (2021).</li><li>De Groote, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+new+gateway-compatible+inducible+lentiviral+vector+platform+allowing+easy+derivation+of+co-transduced+cells.">Generation of a new gateway-compatible inducible lentiviral vector platform allowing easy derivation of co-transduced cells.</a> Biotechniques. 60 (5), 252-259 (2016).</li><li>Ali, M., Roback, L., Mocarski, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+1+ICP6+impedes+TNF+receptor+1-induced+necrosome+assembly+during+compartmentalization+to+detergent-resistant+membrane+vesicles.">Herpes simplex virus 1 ICP6 impedes TNF receptor 1-induced necrosome assembly during compartmentalization to detergent-resistant membrane vesicles.</a> The Journal of Biological Chemistry. 294 (3), 991-1004 (2019).</li><li>Mandal, P., 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=RIP3+induces+apoptosis+independent+of+pronecrotic+kinase+activity.">RIP3 induces apoptosis independent of pronecrotic kinase activity.</a> Moleular Cell. 56 (4), 481-495 (2014).</li><li>Zhang, T., 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=Influenza+virus+Z-RNAs+induce+ZBP1-mediated+necroptosis.">Influenza virus Z-RNAs induce ZBP1-mediated necroptosis.</a> Cell. 180 (6), 1115-1129 (2020).</li><li>Garnish, S. E., 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=Conformational+interconversion+of+MLKL+and+disengagement+from+RIPK3+precede+cell+death+by+necroptosis.">Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis.</a> Nature Communications. 12 (1), 2211 (2021).</li><li>Clay, H., Ramakrishnan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+fluorescent+in+situ+hybridization+in+zebrafish+embryos+using+tyramide+signal+amplification.">Multiplex fluorescent in situ hybridization in zebrafish embryos using tyramide signal amplification.</a> Zebrafish. 2 (2), 105-111 (2005).</li><li>Parra, E. R., 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=Procedural+requirements+and+recommendations+for+multiplex+immunofluorescence+tyramide+signal+amplification+assays+to+support+translational+oncology+studies.">Procedural requirements and recommendations for multiplex immunofluorescence tyramide signal amplification assays to support translational oncology studies.</a> Cancers. 12 (2), 255 (2020).</li></ol>

The authors have nothing to disclose.

This immunofluorescent staining protocol describes the use of tyramide signal amplification (TSA) to increase the sensitivity for signaling events of the human necroptotic signaling pathway that are difficult to detect, including the phosphorylation of RIPK3 and MLKL26. The inclusion of a TSA step significantly improves the detection threshold of p-RIPK3 (S227) and p-MLKL (S358) and increases the sensitivity of p-MLKL (S358) straining. TSA revealed a p-RIPK3 (S227) signal already present in the mo...

Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) are central regulators of necroptotic cell death1,2. Necroptosis is a lytic and inflammatory form of regulated cell death involved in antiviral immunity and autoinflammation. Necroptosis of virus-infected cells immediately shuts down virus replication. Cell lysis following necroptosis induction also releases damage-associated molecular patterns, which stimulate antiviral immunity3,4. Necroptosis is initiated by the activation of RIPK3 following RIP homotypic interaction motif (RHIM)-mediated interactions with one of three upstream activating molecules: RIPK1 (upon TNF receptor 1 [TNFR1] engagement), TIR-domain-containing adapter-inducing interferon-&#946; (TRIF; upon Toll-like receptor 3 and 4 engagement), or the antiviral nucleic acid sensor Z-DNA binding protein 1 (ZBP1)1,2. Necroptosis signaling proceeds through a series of phosphorylation events beginning with the autophosphorylation of RIPK3. The autophosphorylation of human RIPK3 at serine (S)227 inside its kinase domain is a prerequisite for necroptosis by enabling the interaction with MLKL and is commonly used as a biochemical marker for human RIPK3 activation and necroptotic cell death1,5. Once activated, RIPK3 phosphorylates the activation loop of MLKL at threonine (T)357 and S3581. This causes a change in MLKL conformation, resulting in exposure of the N-terminal four helix bundle domain. MLKL then oligomerizes and traffics to the cell membrane where it forms a pore through the insertion of the exposed four helix bundles in the lipid bilayer, eventually leading to cell death2,6.
ZBP1 is an antiviral nucleic acid sensor that recognizes left-handed Z-form nucleic acids including double-stranded RNA in the Z-conformation (Z-RNA). Z-RNA binding occurs via two Z&#945;-domains positioned at the N-terminus of ZBP1. Z-RNA accumulating during RNA and DNA virus infection is thought to directly engage ZBP17,8. Activated ZBP1 recruits RIPK3 through its central RHIMs and induces regulated cell death, including necroptosis9,10. Viruses have adopted numerous escape mechanisms to counteract ZBP1-induced host cell necroptosis11. For example, the herpes simplex virus 1 (HSV-1) ribonucleotide reductase subunit 1, known as ICP6 and encoded by UL39, harbors RHIM at its N-terminus that interferes with ZBP1-mediated RIPK3 activation in human cells12,13,14,15. ZBP1 not only restricts viral replication, but mouse studies have shown that ZBP1 activation causes inflammatory diseases and stimulates cancer immunity16,17,18,19,20,21. Protocols that detect signaling events occurring during ZBP1-induced necroptosis in human cells are, therefore, valuable to assess the role of ZBP1 in these processes.
Tyramide signal amplification (TSA), also referred to as catalyzed reporter deposition (CARD), has been developed to improve the limit of detection and signal-to-noise ratio in antibody-based immunoassays. During TSA, any primary antibody can be used to detect the antigen of interest. Horseradish peroxidase (HRP), coupled to a secondary antibody, catalyzes the local build-up of biotinylated tyramide radicals in the presence of hydrogen peroxide. These activated biotin-tyramide radicals then react with proximal tyrosine residues to form covalent bonds. Potential tyramide-biotin substrates include the antigen itself, the primary and secondary antibodies, and neighboring proteins. Thus, while TSA significantly improves the sensitivity of the assay, some of its spatial resolution is lost. In a final step, biotin molecules are detected using fluorescently labeled streptavidin. The HRP reaction deposits many tyramide-biotin molecules on or near the antigen of interest. This greatly increases the number of streptavidin-fluorochrome binding sites, thereby greatly amplifying the sensitivity of the assay (Figure 1). Alternatively, tyramide can be directly coupled to a fluorochrome, eliminating the need for streptavidin-coupled fluorophores. Protein immunohistochemistry and DNA/RNA in situ hybridization were among the first methods whereby TSA was employed to improve signal intensities22,23. More recently, TSA has been combined with intracellular flow cytometry24 and mass spectrometry25.
Here, we present a protocol to detect serine 227 phosphorylated human RIPK3 (p-RIPK3 [S227]) and phosphorylated human MLKL (p-MLKL [S358]) upon the activation of ZBP1 by HSV-1 infection using immunofluorescence microscopy. We use a necroptosis-sensitive HT-29 human colorectal adenocarcinoma cell line that was transduced to stably express human ZBP1. These cells were infected with an HSV-1 strain expressing a mutant ICP6 protein (HSV-1 ICP6mutRHIM) in which four core amino acids within the viral RHIM (VQCG) were replaced by alanines (AAAA), thereby rendering the ICP6 unable to block ZBP1-mediated necroptosis13,14,15. To overcome the problem of the low signal-to-noise ratio of the currently commercially available antibodies directed against p-RIPK3 and p-MLKL in immunostaining26, we perform a tyramide signal amplification (TSA) step (Figure 1), which results in the robust detection of human p-RIPK3 (S227) and improves the detection sensitivity of human p-MLKL (S358) by an order of magnitude.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit HRP</td>
			<td>Agilent Technologies Belgium</td>
			<td>K4002</td>
			<td>Envision+ System-HRP Labelled Polymer anti-rabbit</td>
		</tr>
		<tr>
			<td>Goat anti-mouse DyLight 633</td>
			<td>Thermofisher</td>
			<td>35513</td>
			<td>Secundary antibody</td>
		</tr>
		<tr>
			<td>HSV-1 ICP0</td>
			<td>Santa Cruz</td>
			<td>sc-53070</td>
			<td>Mouse anti-ICP0(HSV-1) antibody</td>
		</tr>
		<tr>
			<td>IAV-PR8 mouse serum</td>
			<td>In house production</td>
			<td>xx</td>
			<td>Mouse anti-IAV-PR8 polyclonal antibody</td>
		</tr>
		<tr>
			<td>pMLKL</td>
			<td>Abcam</td>
			<td>ab187091</td>
			<td>Rabbit anti-MLKL-phospho S358 antibody</td>
		</tr>
		<tr>
			<td>pRIPK3</td>
			<td>Abcam</td>
			<td>ab209384</td>
			<td>Rabbit anti-RIPK3-phospho S227 antibody</td>
		</tr>
		<tr>
			<td>Fluorophores</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermofisher</td>
			<td>D21490</td>
			<td>To visualise the nucleus of the cells</td>
		</tr>
		<tr>
			<td>Streptavidin coupled to Alexa Fluor 568</td>
			<td>Thermofisher</td>
			<td>S11226</td>
			<td>To visulalise biotin molecules</td>
		</tr>
		<tr>
			<td>Compounds</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30% H2O2</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td>Oxidising substrate, necessary for HRP activity</td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td>SANBIO</td>
			<td>AR1068</td>
			<td>To fix/crosslink the cells</td>
		</tr>
		<tr>
			<td>Biotinyl-tyramide</td>
			<td>R&#38;D Systems</td>
			<td>6241</td>
			<td>To amplify signal, HRP substrate</td>
		</tr>
		<tr>
			<td>BV-6</td>
			<td>Selleckchem</td>
			<td>S7597</td>
			<td>BV6 IAP Inhibitor</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>For cell culture: to detach the cells</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>8.0 g/L NaCl</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>0.4 g/L disodium salt of EDTA</td>
		</tr>
		<tr>
			<td>EDTA 0.04%</td>
			<td>In house formulation</td>
			<td></td>
			<td>1.1 g/L Na2HPO4</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>0.2 g/L NaH2PO4</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>0.2 g/L KCl</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>0.2 g/L Glucose</td>
		</tr>
		<tr>
			<td>Fetal Bovine serum</td>
			<td>TICO</td>
			<td>FBS EU XXX</td>
			<td>For cell culture, maintaining cell culture; lot number: 90439</td>
		</tr>
		<tr>
			<td>GSK&#39;840</td>
			<td>Aobious</td>
			<td>AOB0917</td>
			<td>RIPK3 kinase inhibitor</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td>For cell culture, maintaining cell culture</td>
		</tr>
		<tr>
			<td>MAXblock</td>
			<td>Active Motif</td>
			<td>15252</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10444402</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>S8636</td>
			<td>For cell culture, maintaining cell culture</td>
		</tr>
		<tr>
			<td>TNF-&#945;</td>
			<td>In house production</td>
			<td>-</td>
			<td>Signaling molecule, able to trigger cell death in combination with BV6 and zVAD</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787-50ML</td>
			<td>To permeabilise the cells</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Merck</td>
			<td>11732</td>
			<td>For cell counting, used as live/dead marker at 0,1%</td>
		</tr>
		<tr>
			<td>Trypsine</td>
			<td>Sigma-Aldrich</td>
			<td>T4424</td>
			<td>For cell culture: to detach the cells</td>
		</tr>
		<tr>
			<td>zVAD</td>
			<td>Bachem</td>
			<td>BACE4026865.0005</td>
			<td>Z-Val-Ala-DL-Asp-fluoromethylketone</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Slide 8 well high glass bottom</td>
			<td>iBidi</td>
			<td>80807</td>
			<td>To culture the cells</td>
		</tr>
		<tr>
			<td>Cotton Preping Balls-size medium</td>
			<td>Electron Microscopy Sciences</td>
			<td>71001-10</td>
			<td>To clean the objectives</td>
		</tr>
		<tr>
			<td>Immersol 518 F / 30 &#176;C</td>
			<td>ZEISS</td>
			<td>444970-9000-000</td>
			<td>To visualise the sample at high magnifications</td>
		</tr>
		<tr>
			<td>Lens Cleaner</td>
			<td>ZEISS</td>
			<td>000000-0105-200</td>
			<td>To clean the objectives</td>
		</tr>
		<tr>
			<td>LSM880 Fast Airyscan confocal microscope</td>
			<td></td>
			<td></td>
			<td>To visualise the sample</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Office</td>
			<td>xx</td>
			<td>To process the data</td>
		</tr>
		<tr>
			<td>Prism 9</td>
			<td>Graphpad</td>
			<td>xx</td>
			<td>To analyse the data- statistical testing and graph generation</td>
		</tr>
		<tr>
			<td>Volocity 6.3</td>
			<td>Volocity</td>
			<td>xx</td>
			<td>To perform quantifications</td>
		</tr>
		<tr>
			<td>Zen black</td>
			<td>ZEISS</td>
			<td>xx</td>
			<td>To aquire and process images</td>
		</tr>
		<tr>
			<td>Zen blue</td>
			<td>ZEISS</td>
			<td>xx</td>
			<td>To visualise images</td>
		</tr>
		<tr>
			<td>Viruses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HSV-1 (mutRHIM) F strain</td>
			<td>produced by&#160; Dr. Jiahuai Han</td>
			<td>in house replication</td>
			<td>HSV-1 as a trigger for necroptosis; RHIM core domain of UL39/ICP6 is mutated (VQCG&#62;AAAA)</td>
		</tr>
		<tr>
			<td>HSV-1 (WT) F strain</td>
			<td>Produced by Dr. Jiahuai Han</td>
			<td>in house replication</td>
			<td>HSV-1 (WT) as a negative control for necroptosis induction (ICP6 inhibition)</td>
		</tr>
		<tr>
			<td>IAV PR8</td>
			<td>in house stock</td>
			<td>in house replication</td>
			<td>IAV as a trigger for necroptosis</td>
		</tr>
	</tbody>
</table>

tyramide signal amplification for the immunofluorescent staining of zbp1-dependent phosphorylation of ripk3 and mlkl after hsv-1 infection in human cells

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

The immunofluorescent detection of MLKL phosphorylation and especially RIPK3 phosphorylation in human cells is technically challenging26. We here present an improved staining protocol for human p-RIPK3 (S227) and p-MLKL (S358) upon the activation of ZBP1. The protocol includes a TSA step to improve the detection limit and sensitivity of the fluorescent signals. To validate the method, a side-by-side comparison of the TSA-mediated immunofluorescence with standard indirect fluorescent staining of bo...

Watch this Scientific Journal Video about Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells at J

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical ...

tyramide-signal-amplification-for-immunofluorescent-staining-zbp1

Research

JoVE Journal

Immunology and Infection

2.0K Views.  VIB-UGent Center for Inflammation Research. Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.

Video: Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...