We thank Barbara Jakli&#269; for&#160;technical assistance. This research was financially supported by the Slovenian Research and Innovation Agency (research core funding no. P4-0165 and project Z4-3217: Deciphering redox-related signaling interconnectedness in potato resistance against viruses).

NOTE: Sections 1 and 2 describe a modified protocol for plant material preparation based on the methods outlined by Lukan et al.7. Specifically, some modifications to controlled environmental conditions and inoculum preparation were made.
1. Growing potato plants
<ol>
	<li>Grow healthy potato cv. Rywal plants in stem node tissue culture8.</li>
	<li>After 6-8 weeks, cut ten 1 cm-long explants containing nodes from potato plants on sterile paper under sterile conditions using sterile tweezers and a scalpel knife.</li>
	<li>Transfer them to plastic boxes with Murashige and Skoog (MS30) medium (Figure 1A) and grow them under controlled environmental conditions (22 &#176;C in the light and 19 &#176;C in the dark with 250 &#181;E or &#181;mol/m2/s&#160;radiation and a 16-h photoperiod, at a relative humidity of 55% &#177; 5%).</li>
	<li>Transfer them to the soil 2 weeks after micropropagation.
	<ol>
		<li>Fill the pots (r = 10 cm) with soil. Use a finger to create a 3-4 cm deep hole in the soil in the middle of a pot, fill the hole with water, and wait for it to absorb.</li>
		<li>Place one plant per pot into the hole, leaving the leaves above the surface. Gently cover the roots with soil (Figure 1B). 
		NOTE: Some plants might not develop roots. Exclude those plants from the analysis.</li>
	</ol>
	</li>
	<li>Grow the plants in soil in a growth chamber under controlled environmental conditions (22 &#176;C in the light and 19 &#176;C in the dark, at a relative humidity of 55% &#177; 5%, with 250 &#181;mol/m2/s&#160;radiation and a 16-h photoperiod).</li>
	<li>After 3-4 weeks, plants are ready. Use these plants to be inoculated. 
	NOTE: Plants should have at least 3-4 fully developed leaves with visible leaflets (Figure 1C). They should look healthy, with no visible symptoms (yellow or brown leaves), which could be mistaken for lesions caused by PVY. To achieve comparable results, plants should be inoculated at the same time (e.g., at 9 am) in all experiments to avoid possible effects of circadian rhythm on plant immune response and lesion formation9,10,11.</li>
</ol>
2. Inoculum preparation and potato inoculation
<ol>
	<li>Inoculate the plant with PVY strain N-Wilga&#160;(PVYN-Wi; accession no. EF558545) inoculum as stated below. 
	NOTE: More plants can be inoculated in parallel if more than one digital microscope is available. Plants should have at least three fully developed leaves before inoculation (Figure 1C). PVYN-Wi is multiplied and maintained in potato cv. Pentland.
	<ol>
		<li>Prepare phosphate buffer, supplemented with sodium diethyldithiocarbamate (DIECA), for inoculation: Mix 1.3 mL of 0.2 M NaH2PO4, 8.7 mL of 0.2 M Na2HPO4, and 0.225 g of DIECA. Make up the volume to 100 mL using ddH20. Adjust pH to 7.6 by adding 1 M NaOH or 1 M HCl solution.</li>
		<li>Harvest 6-8 weeks old PVYN-Wi infected potato cv. Pentland plants from tissue culture in extraction bags with filter net (0.5 g per 6 plants) and add phosphate buffer supplemented with DIECA (4x mass of plant material, mass: volume ratio). Use a hand homogenizer for 1-2 min to obtain a homogeneous solution.</li>
		<li>Lightly dust the first three bottom fully developed leaves of 3-4 weeks old potato plants (from step 1.6) with carborundum powder. 
		NOTE: Adjust the amount of carborundum powder. Too much may cause damage, while too little might result in ineffective infection. Optimally, 0.1 mg/cm of carborundum powder is used, which is approximately 1.5 mg of powder per leaf of average size (approximately 15 cm2).</li>
		<li>Gently rub the leaves with the inoculum (~100 &#181;L per leaf). After 10 min, thoroughly wash the leaves with tap water.
		<ol>
			<li>Be careful not to harm the leaves. Do not exceed the incubation time. Adjust the amount of the inoculum according to leaf size - 6.5 &#181;L/cm, which is approximately 100 &#181;L per leaf of average size (approximately 15 cm2).</li>
		</ol>
		</li>
		<li>Transfer the plants to a growth chamber and grow them under controlled environmental conditions (28 &#176;C in the light and 28 &#176;C in the dark at a relative humidity of 55% &#177; 5%, with 250 &#181;mol/m2/s&#160;radiation and a 16 h photoperiod) for 3 days.</li>
	</ol>
	</li>
</ol>
3. Plant preparation and using the digital microscope for recording lesion development
<ol>
	<li>Transfer the inoculated plants from the growth chamber maintained at 28 &#176;C to a growth chamber at 22 &#176;C 3 days after inoculation, with other conditions as described in step 1.5.</li>
	<li>Select a plant for observation. Immobilize the second inoculated leaf using tape (Figure 1D). Make sure that the desired leaf is fully immobilized before starting with imaging (Figure 1D).</li>
	<li>Install a software application for image capture on the laptop computer. Connect the digital microscope to the computer. Open the software. 
	NOTE: Make sure that the plant, digital microscope, and laptop are positioned on the shelf near the socket to plug in the computer. The decision on the selection of leaves to be monitored might depend on the biological question studied, e.g., the type of process that leads to the formation of limited programmed cell death.</li>
	<li>Adjust the microscope above the immobilized leaf. Focus using the dial on the digital microscope (shown in Figure 1E).
	<ol>
		<li>Ensure that the line of sight is as wide as possible to increase the chances of capturing the formation of the lesion but still magnified enough to spot the lesion&#39;s appearance (typically, 25x magnification is used).</li>
	</ol>
	</li>
	<li>Set the camera settings. Click on the Settings button in the top right corner of the image (Figure 2A, circled part 1) and adjust camera settings: Brightness (60-70), Contrast (10-15), Hue (0), White Balance, Saturation (45-50), Sharpness (0), Gamma (5) (Figure 2B). The settings used in this study were as follows: Brightness (64), Contrast (14), Hue (0), Saturation (47), Sharpness (0), Gamma (5). 
	NOTE: Camera settings should be adjusted to outside light to ensure the best image quality. Settings could be adapted according to the user&#39;s conditions in the growth chamber.</li>
	<li>Set the image capture settings by clicking on the icon for Image Capture (Figure 2C, circled part 2). Click on the button Time-Lapsed Video and set Image Capture every 15 min for 24 h (Figure 2C). 
	NOTE: The interval between the images taken and imaging duration are fully flexible and could be adapted according to the needs of the experiment. In the time between image capture, the LED light is switched off. The LED light is automatically switched on during image capture.</li>
	<li>Start image capture by clicking on the START button (Figure 2C). 
	NOTE: After being transferred from 28 &#176;C to 22 &#176;C, plants should start to develop lesions within 24 h. If not, this could be a sign of ineffective inoculation. Adjust the amount of carborundum powder, ensure that the time of inoculum incubation on leaves was correct, and determine virus abundance in the inoculum by qPCR.</li>
	<li>To save images, select all images and click the Save icon (Figure 2B, circled part 3). Set Export Options. Set DPI at maximum (300) (Figure 2D). After saving, delete all pictures in the program.</li>
	<li>For image analysis, any program for image viewing/editing is sufficient. The usage of free software for image editing, ImageJ, is explained below.
	<ol>
		<li>Import the time sequence of images from a single field of vision (on the top left corner, click File, select Import, and then Image Sequence). Paste the path of the directory of saved pictures and press the OK button to start the conversion.</li>
		<li>After the conversion, the software automatically opens an internal video player showing the final video. Export the video file by clicking on File &#62; Save as option and selecting AVI Format. A small window opens. Set the Frame Rate as 0.3 fps and press OK to save the video as an AVI video file. 
		NOTE: The time of lesion development can also be determined by manually checking all of the images and finding an image where the lesion appears.</li>
	</ol>
	</li>
</ol>

Quantifying Cell Death Initiation Through Continuous Imaging

<ol><li>Karasev, A. V., Gray, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+and+emerging+challenges+of+potato+virus+y+in+potato%5BTitle%5D)+AND+%22Annual+Review+of+Phytopathology%22%5BJournal%5D)">Continuous and emerging challenges of potato virus y in potato.</a> Annual Review of Phytopathology. 51, 571-586 (2013).</li>
<li>Quenouille, J., Vassilakos, N., Moury, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potato+virus+Y%3A+A+major+crop+pathogen+that+has+provided+major+insights+into+the+evolution+of+viral+pathogenicity%5BTitle%5D)+AND+%22Molecular+Plant+Pathology%22%5BJournal%5D)">Potato virus Y: A major crop pathogen that has provided major insights into the evolution of viral pathogenicity.</a> Molecular Plant Pathology. 14 (5), 439-452 (2013).</li>
<li>Szajko, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+novel+gene+Ny-1+on+potato+chromosome+IX+confers+hypersensitive+resistance+to+Potato+virus+Y+and+is+an+alternative+to+Ry+genes+in+potato+breeding+for+PVY+resistance%5BTitle%5D)+AND+%22Theoretical+and+Applied+Genetics%22%5BJournal%5D)">The novel gene Ny-1 on potato chromosome IX confers hypersensitive resistance to Potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance.</a> Theoretical and Applied Genetics. 116 (2), 297-303 (2008).</li>
<li>Szajko, K., Strzelczyk-Żyta, D., Marczewski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ny-1+and+Ny-2+genes+conferring+hypersensitive+response+to+potato+virus+Y+%28PVY%29+in+cultivated+potatoes%3A+Mapping+and+marker-assisted+selection+validation+for+PVY+resistance+in+potato+breeding%5BTitle%5D)+AND+%22Molecular+Breeding%22%5BJournal%5D)">Ny-1 and Ny-2 genes conferring hypersensitive response to potato virus Y (PVY) in cultivated potatoes: Mapping and marker-assisted selection validation for PVY resistance in potato breeding.</a> Molecular Breeding. 34 (1), 267-271 (2014).</li>
<li>Lukan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+death+is+not+sufficient+for+the+restriction+of+potato+virus+Y+spread+in+hypersensitive+response-conferred+resistance+in+potato%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">Cell death is not sufficient for the restriction of potato virus Y spread in hypersensitive response-conferred resistance in potato.</a> Frontiers in Plant Science. 9, 168(2018).</li>
<li>Baebler, Š, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Salicylic+acid+is+an+indispensable+component+of+the+Ny-1+resistance-gene-mediated+response+against+Potato+virus+y+infection+in+potato%5BTitle%5D)+AND+%22Journal+of+Experimental+Botany%22%5BJournal%5D)">Salicylic acid is an indispensable component of the Ny-1 resistance-gene-mediated response against Potato virus y infection in potato.</a> Journal of Experimental Botany. 65 (4), 1095-1109 (2014).</li>
<li>Lukan, T., Coll, A., Baebler, Š, Gruden, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+virus+spread+around+the+cell+death+zone+at+spatiotemporal+resolution+using+confocal+microscopy%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Analysis of virus spread around the cell death zone at spatiotemporal resolution using confocal microscopy.</a> Methods in Molecular Biology. 2447, 261-270 (2022).</li>
<li>Vinterhalter, D., Dragiüeviü, I., Vinterhalter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potato+in+vitro+culture+techniques+and+biotechnology%5BTitle%5D)+AND+%22Fruit%2C+Vegetable+and+Cereal+Science+and+Biotechnology%22%5BJournal%5D)">Potato in vitro culture techniques and biotechnology.</a> Fruit, Vegetable and Cereal Science and Biotechnology. 2, Special Issue 16-45 (2008).</li>
<li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Timing+of+plant+immune+responses+by+a+central+circadian+regulator%29%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Timing of plant immune responses by a central circadian regulator).</a> Nature. 470, 110-115 (2011).</li>
<li>Roden, L. C., Ingle, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lights%2C+rhythms%2C+infection%3A+The+role+of+light+and+the+circadian+clock+in+determining+the+outcome+of+plant-pathogen+interactions%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Lights, rhythms, infection: The role of light and the circadian clock in determining the outcome of plant-pathogen interactions.</a> Plant Cell. 21 (9), 2546-2552 (2009).</li>
<li>Srivastava, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+circadian+rhythm+in+plant+system%3A+An+update+from+development+to+stress+response%5BTitle%5D)+AND+%22Environmental+and+Experimental+Botany%22%5BJournal%5D)">Role of circadian rhythm in plant system: An update from development to stress response.</a> Environmental and Experimental Botany. 162, 256-271 (2019).</li>
<li>Mulaosmanovic, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+method+for+detection+and+quantification+of+lesions+on+leaf+scale+based+on+trypan+blue+staining+and+digital+image+analysis%5BTitle%5D)+AND+%22Plant+Methods%22%5BJournal%5D)">High-throughput method for detection and quantification of lesions on leaf scale based on trypan blue staining and digital image analysis.</a> Plant Methods. 16, 62(2020).</li>
<li>Martinelli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+methods+of+plant+disease+detection.+A+review%5BTitle%5D)+AND+%22Agronomy+for+Sustainable+Development%22%5BJournal%5D)">Advanced methods of plant disease detection. A review.</a> Agronomy for Sustainable Development. 35, 1-25 (2015).</li>
<li>Ali, M., Bachik, N., Muhadi, N. A., Tuan Yusof, T. N., Gomes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-destructive+techniques+of+detecting+plant+diseases%3A+A+review%5BTitle%5D)+AND+%22Physiological+and+Molecular+Plant+Pathology%22%5BJournal%5D)">Non-destructive techniques of detecting plant diseases: A review.</a> Physiological and Molecular Plant Pathology. 108, 101426(2019).</li>
<li>Sankaran, S., Mishra, A., Ehsani, R., Davis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+advanced+techniques+for+detecting+plant+diseases%5BTitle%5D)+AND+%22Computers+and+Electronics+in+Agriculture%22%5BJournal%5D)">A review of advanced techniques for detecting plant diseases.</a> Computers and Electronics in Agriculture. 72 (1), 1-13 (2010).</li>
<li>Rowarth, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA-Seq+analysis+reveals+potential+regulators+of+programmed+cell+death+and+leaf+remodelling+in+lace+plant+%28Aponogeton+madagascariensis%29%5BTitle%5D)+AND+%22BMC+Plant+Biology%22%5BJournal%5D)">RNA-Seq analysis reveals potential regulators of programmed cell death and leaf remodelling in lace plant (Aponogeton madagascariensis).</a> BMC Plant Biology. 21 (1), 375(2021).</li>
</ol>

The authors declare that they have no conflicts of interest.

The demonstrated protocol allows the user to accurately determine the cell death initiation rate by continuously imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope. Even though there are numerous ways of monitoring lesion and plant disease occurrence12,13,14,15, this protocol presents the advantage of light-independent measuring without disturbing the plant&#39;s circadian rhythm, as the light is switched off between measurements.
After the inoculation, plants should grow at 28 &#176;C for 3 days. Ny-1 resistance gene, which induces a hypersensitive response, is temperature-dependent, and in plants grown at higher temperatures, leads to abortion of resistance, which is manifested as lack of lesion formation and systemic virus spread3. After plants are transferred to 22 &#176;C, cell death is initiated, so for accurate results, observation with a digital microscope should begin as soon as possible after this transfer. Another crucial step in the preparation of the plant for imaging is the immobilization of the leaf (Figure 1D), as the plant will continue to grow during imaging, which could move the observed leaf out of focus, or such set-up will not give desired results.
If the described protocol is used on transgenic plants with altered components of interest, hypothesized to be involved in cell death initiation, the protocol enables the user to determine if the decreased level of a studied component affects the rate of cell death initiation. By that, components involved in cell death initiation can be identified in pathosystems, where programmed cell death occurs, using this protocol. Other methods for identifying these components are, for example, transcriptomic analysis such as RNA-seq&#160;or various forms of microscopy, which can be expensive and time-consuming16. The method described in this protocol allows for easy and inexpensive identification of components involved in cell death initiation by observing differences in cell death initiation rates between transgenic and control plants. Optimally, in such a set-up, two digital cameras must be used, as a transgenic plant should be analyzed in parallel with a control plant within the same experiment.
In this protocol, PVY strain N-Wilga was used; however, other strains of this virus, for example, GFP-tagged PVY (PVY-N605(123)-GFP)7, could also be used. Moreover, other pathosystems, which result in programmed cell death development could be studied using this protocol with minor modification.

Potato is one of the world&#39;s most widely grown food crops, fourth place behind rice, wheat, and corn. However, potato production can be severely affected by potato virus Y (PVY), which is currently considered its most important virus pathogen1,2. In potato plants cv. Rywal, several strains of PVY (including PVY strain N-Wilga) trigger hypersensitive response (HR)-conferred resistance, where the pathogen&#39;s restriction to the infection site manifests as necrotic lesions on inoculated leaves3. In this pathosystem, HR is mediated by Ny-1 resistance gene, which is temperature-dependent, as plants grown at lower temperatures efficiently develop necrotic lesions, whereas in plants grown constitutively at elevated (28 &#176;C) temperature, abortion of resistance is demonstrated as lack of lesion formation and systemic virus spread3,4. When the plants are transferred to a lower temperature (22 &#176;C), cell death is initiated, which can be exploited to follow the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance.
This protocol demonstrates a simple method for the determination of cell death initiation rate using a digital microscope. By imaging the inoculated leaves after transferring the plant from 28 &#176;C to 22 &#176;C, a digital microscope enables continuous observation of the leaf in desired intervals. Unlike the use of other methods (for example, confocal microscopy or observation of lesion formation with the naked eye), this allows determination of the exact time of lesion formation and therefore, the cell death initiation rate up to minutes exactly, as opposed to hours in aforementioned methods5,6. Digital microscope use is also independent of light and can therefore be used during day and night. This protocol can also be used to identify components involved in cell death initiation or to determine the effects of different components on cell death initiation rate if used plants are transgenic and have altered levels of components of interest.

<table><tbody><tr><td>Alcohol burner</td><td>Mikro+Polo</td><td>SH-234002455</td><td>For tweezers and scalpel sterilization</td></tr><tr><td>Autoclave A-21 CAV</td><td>Kambi&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/65824/65824eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</td><td>N/A</td><td></td></tr><tr><td>Bacto Agar</td><td>Becton, Dickinson and Company</td><td>214010</td><td></td></tr><tr><td>Carborundum powder</td><td>VWR Chemicals</td><td>22505297</td><td></td></tr><tr><td>DinoCapture 2.0&amp;nbsp;</td><td>Dino-Lite</td><td>Version 2.0</td><td>software for digital microscope</td></tr><tr><td>Dino-Lite Edge AM7915MZTL digital microscope</td><td>AnMo Electronics Corporation</td><td>AM7915MZTL</td><td></td></tr><tr><td>Ethanol, 70%</td><td>Stella Tech</td><td>P94000</td><td>For tweezers and scalpel sterilization</td></tr><tr><td>Extraction bags</td><td>Bioreba</td><td>420100</td><td></td></tr><tr><td>Growth chamber FS-WI</td><td>Photon Systems Insturments</td><td>N/A</td><td></td></tr><tr><td>Hand homogenizer</td><td>Bioreba</td><td>400010</td><td></td></tr><tr><td>Hawita Special Substrate</td><td>HAWITA Gruppe</td><td>2000000071701</td><td>Ready to use substrate, made using peat (H4-H6 and H6-H8)</td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Merck</td><td>109057</td><td></td></tr><tr><td>Label tape</td><td>Sigma</td><td>L8144-5EA</td><td></td></tr><tr><td>Laptop computer with installed DinoCapture 2.0&amp;nbsp;</td><td>HP</td><td>Z2V77EA#BED</td><td>Computer needs to be transferable as experiment takes part in a growth chamber</td></tr><tr><td>Murashige and Skoog medium</td><td>Duchefa Biochemie</td><td>M02220100</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Emsure</td><td>1065860500</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Emsure</td><td>1064700250</td><td></td></tr><tr><td>Pasteur pipette&amp;nbsp; 0.5 mL</td><td>Brand</td><td>21500209</td><td></td></tr><tr><td>pH-meter</td><td>Mettler Toledo</td><td>ML1601</td><td></td></tr><tr><td>Plastic boxes&amp;nbsp;</td><td>Cvetlice Dornig</td><td>VCG10.5</td><td>Radius = 10.5 cm</td></tr><tr><td>Plastic pots&amp;nbsp;</td><td>Lab Associates</td><td>DIS40003</td><td>Radius = 11.5 cm (top), Radius = 9.8 cm (bottom)</td></tr><tr><td>Saccharose</td><td>Kemika d.d.</td><td>1800408</td><td></td></tr><tr><td>Sodium Diethyldithiocarbamate (DIECA)</td><td>Sigma-Aldeich</td><td>228680</td><td>Sodium diethyldithiocarbamate trihydrate, ACS reagent</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Merck</td><td>106462</td><td></td></tr><tr><td>Sterile surgical blades</td><td>Braun</td><td>4511733633</td><td></td></tr><tr><td>Tweezers</td><td>Braun</td><td>BD033R</td><td></td></tr></tbody></table>

studying cell death initiation using a digital microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

This study demonstrates a step-by-step protocol for studying cell death initiation through lesion occurrence on potato cv. Rywal, with a digital microscope. This enables determining the exact time of programmed cell death initiation.
Plants that have developed roots were put in soil 2 weeks after potato cv. Rywal micropropagation (Figure 1A,B). After 3-4 weeks of growth under described conditions, plants with at least 3-4 fully developed leaves with visible leaflets that looked healthy, with no signs of abscission, were used for further analysis (Figure 1C). Using a digital microscope as described in this protocol, we observed the same area on the inoculated leaf at 15-min-intervals and determined the lesion occurrence and expansion in time (Figure 3). The lesion occurred at 15 h 30 min (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65824/65824fig01.jpg" /> 
Figure 1: Plant preparation for analysis with a digital microscope. (A) A plastic box with MS 30 medium and potato cv. Rywal plant explants containing nodes. (B) Potato cv. Rywal plant in soil (2 weeks after micropropagation). (C) Potato cv. Rywal plant, ready for inoculation (4 weeks after being put in soil), having at least three fully developed leaves. (D) Second inoculated leaf (arrow) of potato cv. Rywal plant positioned and immobilized (arrow) with tape. (E) Plant positioned under the digital microscope with the arrow pointing to the dial used for focusing. <a href="https://www.jove.com/files/ftp_upload/65824/65824fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65824/65824fig02.jpg" /> 
Figure 2: Digital software setting for recording lesion development. (A) Software interface - circled with red are options for the button for (1) camera&#160;settings, (2) image&#160;capture&#160;settings, and (3) saving&#160;images. (B) Window with camera settings, which opens with a click on (1) in panel A. Brightness, Contrast, Saturation, Sharpness, and Gamma should be properly adjusted. (C) Window with image capture settings, which opens with a click on (2) denoted in panel A. (D) Window with image saving settings, which opens with a click on (3) denoted in panel A. <a href="https://www.jove.com/files/ftp_upload/65824/65824fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65824/65824fig03.jpg" /> 
Figure 3: Lesion formation on the inoculated leaf observed under the digital microscope. Images of the central part of PVY-inoculated potato leaf at 23.6x magnification as seen under the digital microscope, taken at intervals of 5 min. Inoculated plants were put at 28 &#176;C for 3 days, and on the third day, the observation with a digital microscope at 22 &#176;C started at 7:00. (A) At 21:02, the lesion is not yet visible, (B) 90 min later, at 22:32, the lesion is visible. (C) The lesion expansion was observed at 01:02 and (D) 07:32 the next morning. Experiment was repeated two times, and the lesions occurred 8 h 15 min, and 12 h after cell death initiation, respectively. <a href="https://www.jove.com/files/ftp_upload/65824/65824fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Author Spotlight: A Streamlined Approach to Studying Cell Death Initiation in Hypersensitive Response 

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

Studying Cell Death Initiation Using a Digital Microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as ...

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1.4K Views.  National Institute of Biology. Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction. 

Video: Author Spotlight: A Streamlined Approach to Studying Cell Death Initiation in Hypersensitive Response 

One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. Here, we describe a quick and cost-effective one-step assay for simultaneously assessing the major modes of clonogenic cell death induced by IR, i.e., apoptosis, mitotic catastrophe, and cellular senescence. In this method, cells grown on a cover slip are irradiated with X-rays and stained with 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI). Using fluorescence microscopy, apoptosis, mitotic catastrophe, and cellular senescence are identified based on the characteristic morphologies of the DAPI-stained nuclei. Apoptosis is determined by the presence of apoptotic bodies (i.e., condensed and fragmented nuclei). Mitotic catastrophe is determined by the presence of nuclei that exhibit two or more distinct lobes and micronuclei. Cellular senescence is determined by the presence of senescence-associated heterochromatic foci (i.e., nuclear DNA containing 30-50 bright, dense foci). This approach allows the experimenter to easily screen for clonogenic cell death modes using various cell lines, treatment settings, and/or time points, with the goal of elucidating the mechanisms of cell death in the target cells and conditions of interest.

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effects of IR on malignant tumors and normal tissues. Here, we describe a one-step assay for assessing the major modes of IR-induced clonogenic cell death based on morphology of 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nuclei visualized by fluorescence microscopy.

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. ...

Cancer Research

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Medicine

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to assess factors that influence neuronal fate. These assays require the observation of separate populations of cells at discrete time points. As a result, individual cells cannot be followed prospectively over time, severely limiting the ability to discriminate whether subcellular events, such as puncta formation or protein mislocalization, are pathogenic drivers of disease, homeostatic responses, or merely coincidental phenomena. Single-cell longitudinal microscopy overcomes these limitations, allowing the researcher to determine differences in survival between populations and draw causal relationships with enhanced sensitivity. This video guide will outline a representative workflow for experiments measuring single-cell survival of rat primary cortical neurons expressing a fluorescent protein marker. The viewer will learn how to achieve high-efficiency transfections, collect and process images enabling the prospective tracking of individual cells, and compare the relative survival of neuronal populations using Cox proportional hazards analysis.

Here, we present a protocol to monitor survival on a single-cell basis and identify variables that significantly predict cell death.

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to ...

Neuroscience

A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to study the process of neuronal survival upon cytotoxic stress. Most methods are based on biochemical approaches that do not allow single-cell resolution or involve complex and costly methodologies. Presented here is a versatile, inexpensive, and effective experimental paradigm to study neuronal survival. This method takes advantage of sparse fluorescent labeling of the neurons followed by live imaging and automated quantification. To this aim, the neurons are electroporated to express fluorescent markers and co-cultured with non-electroporated neurons to easily regulate cell density and increase survival. 
Sparse labeling by electroporation allows a simple and robust automated quantification. In addition, fluorescent labeling can be combined with the co-expression of a gene of interest to study specific molecular pathways. Here, we present a model of stroke as a neurotoxic model, namely, the oxygen-glucose deprivation (OGD) assay, which was performed in an affordable and robust homemade hypoxic chamber. Finally, two different workflows are described using IN Cell Analyzer 2200 or the open-source ImageJ for image analysis for semi-automatic data processing. This workflow can be easily adapted to different experimental models of toxicity and scaled up for high-throughput screening. In conclusion, the described protocol provides an approachable, affordable, and effective in vitro model of neurotoxicity, which can be suitable for testing the roles of specific genes and pathways in live imaging and for high-throughput drug screening.

Here, we present a simple, potent, and versatile methodology to investigate neuronal survival upon cytotoxic stress in primary cortical neurons with cellular resolution in real time or in fixed material.

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and ...

Quantifying Cell Death in Human Colonoids in Response to Cytotoxic Perturbagens

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-&#945; and IFN-&#947;, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.

Author Spotlight: Understanding Cytokine-Induced Cell Death in Intestinal Epithelial Cells Using Human Organoids

This protocol describes a simple and cost-effective method to investigate and quantify cell death in human colonic organoids in response to cytotoxic perturbagens such as cytokines. The approach employs a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software to quantify single-organoid responses to cytotoxic stimuli.

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease ...

An Introduction to Cell Death

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as cell injury, low nutrient levels, or signaling proteins. Whereas necrosis is considered to be an &#8220;accidental&#8221; or unexpected form of cell death, evidence exists that apoptosis and autophagy are both programmed and &#8220;planned&#8221; by cells.In this introductory video, JoVE highlights key discoveries pertaining to cell death, including recent work done in worms that helped identify genes involved in apoptosis. We then explore questions asked by scientists studying cell death, some of which look at different death pathways and their interactions. Finally, several methods to assess cell death are discussed, and we note how researchers are applying these techniques in their experiments today.

Cell Death: Apoptosis, Autophagy and Necrosis

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as ...