We thank Darren Obbard for Drosophila image in Figure 3C and in video manuscript; J. Marie Hardwick, Wade Gibson, and Heather M. Lamb for valuable discussion of this manuscript. This work was supported by a Sir Edward Youde Memorial Fellowship (H.L.T.), Dr. Walter Szeto Memorial Scholarship (H.L.T.), Fulbright grant 007-2009 (H.L.T.), Life Science Research Foundation fellowship (H.L.T.), and NCI K22 grant CA204458 (H.L.T.). Ho Lam Tang was a Shurl and Kay Curci Foundation Fellow of the Life Sciences Research Foundation (2014-2017).

1) Preparation of CaspaseTracker Biosensor Flies
<ol>
	<li>Anesthetize flies with CO2, and use a paintbrush to transfer 7 to 10 caspase-sensitive Gal4 (DQVD)19 virgin females and 7 to 10 G-Trace53 Gal4 reporter young male flies (or vice versa) in the same vial with fly food and fresh yeast paste. 
	NOTE: Cross of Caspase-sensitive (DQVD) Gal4 and G-Trace flies will produce CaspaseTracker progeny flies. Cross of Caspase-insensitive (DQVA)19 Gal4 and G-Trace flies will provide negative control flies (see discussion). Fresh yeast paste serves as protein source to enhance egg production, so that increases number of progeny. Select virgin females and young male flies according to their phenotypes54.</li>
	<li>Incubate the flies at 18 degrees Celsius (oC) during the cross for 3 to 7 days, and then transfer the flies to a new vial to set up a new cross at 18 &#176;C. Continue to incubate the original vial at 18 &#176;C until progeny flies eclose. 
	NOTE: Transfer the parent flies to new vials to avoid overcrowding of progeny at the original vial. Parent flies can produce progeny with fresh food and yeast paste at the first 2 to 3 switches, and then the productivity will significantly decrease with time. Raising flies 18 &#176;C can reduce non-specific signal of CaspaseTracker biosensor (see discussion).</li>
	<li>Select progeny flies with correct phenotypes54 for following experiments. 
	NOTE: The transgenes of both caspase-sensitive Gal4 and G-Trace here are located at the second chromosome, balanced with CyO balancer. Select the non-curly wing progeny (without CyO), which has both transgenes of caspase-sensitive Gal4 and G-Trace.</li>
</ol>
2) Application of Transient Cell Death Induction to CaspaseTracker Biosensor Flies
<ol>
	<li>Transfer 10 to 20 newly eclosed female flies to a new vial with fresh fly food and fresh yeast paste for 1 day at 18 &#176;C to allow egg chamber production by oogenesis. 
	NOTE: Keeping the female with male flies might enhance egg chamber production.</li>
	<li>To induce egg chambers to undergo apoptosis by cold shock, transfer the female flies to new empty vial, which is then placed at -7 &#176;C for 1 h. 
	NOTE: Cold shock injures cells by inducing plasma membrane rupture55,56.</li>
	<li>To induce egg chambers to undergo apoptosis by protein starvation, transfer the female flies to a new vial with 8% sucrose and 1% agar food at 18 &#176;C for 3 days. 
	NOTE: Protein starvation (non-protein food) can trigger egg chambers to undergo apoptosis57,58,59 and autophagy60,61. Switch flies to a new vial with 8% sucrose and 1% agar food every day to keep optimal condition of the sucrose fly food.</li>
	<li>Transfer the stressed flies back to a new vial with fresh fly food and fresh yeast paste for 3 days at 18 &#176;C to allow them to recover. Dissect the starved and the starved-recovered flies to obtain egg chambers at ovaries as described62. 
	NOTE: To dissect Drosophila to obtain ovaries, anesthetize flies with CO2, and use 2 pairs of forceps to remove fly head, and use the forceps to pull the base of the abdomen to remove the ovaries of the flies.</li>
</ol>
3) Fixation and Staining of Dissected Egg Chambers for Imaging
<ol>
	<li>Transfer the dissected egg chambers together with around 0.5 mL phosphate buffered saline (PBS) to 1 mL centrifuge tubes. Allow the eggs to settle down. 
	NOTE: Coat the plastic pipette tips with 1% bovine serum albumin (BSA) dissolved in water or PBS to prevent the egg chambers to stick on the plastic surface of the tips. Perform the following procedures in dark to avoid photobleaching of red fluorescent protein (RFP, also known as DsRed) and green fluorescent protein (GFP) in the egg chambers.</li>
	<li>Remove the PBS by pipetting, and then apply 0.5 mL 4% paraformaldehyde in PBS to fix the egg chambers at room temperature in dark for 20 to 30 min. 
	NOTE: Apply gentle rotation in this and the following incubation steps.</li>
	<li>Remove the paraformaldehyde by pipetting, and then washed the egg chamber with 0.5 mL PBST (PBS + 0.1% Triton X-100) for 3 times. 
	NOTE: Prolonged fixation could reduce the RFP and GFP signals.</li>
	<li>Incubate the egg chambers with PBST for 1 to 2 h&#160;at room temperature or overnight at 4 &#176;C with gentle rotation to permeabilize the egg chambers. 
	NOTE: PBST can also avoid egg chambers to stick to the non-BSA coated plastic surface.</li>
	<li>Remove the PBST by pipetting, and then apply 0.5 mL of 10 &#956;g/mL of blue nuclear Hoechst dye in PBST to egg chambers for 1 to 2 h&#160;at room temperature to stain for nucleus. 
	NOTE1: Avoid prolonged incubation with nuclear dye as this will increase non-specific signal. 
	NOTE2: Alternative approach to stain nucleus without Hoechst is to add 200 &#956;L anti-bleaching mounting agent with DAPI (see materials) and incubate overnight63, before mounting the tissues on glass cover slip as described at protocol 3.8. 
	NOTE3: Perform the staining and the following procedures in dark to avoid photobleaching.</li>
	<li>Remove the nuclear dye by pipetting, and then apply 0.5 mL PBST to wash the egg chambers in the 1 mL centrifuge tubes for 3 times, with 10 to 20 min&#160;incubation with gentle rotation between each washing step.</li>
	<li>Remove all PBST with fine pipette, and then apply 200 &#956;L anti-bleaching mounting agent (see materials) to incubate the egg chambers at room temperature for 3 h&#160;or overnight at 4 &#176;C until the egg chambers sink to the bottom of the tube. 
	NOTE: Tissues that have fully absorbed the mounting agent sink to the bottom of the tube.</li>
	<li>Mount the stained egg chambers by transferring them with 200 &#956;L anti-bleaching mounting agent on a pre-cleaned glass slide for imaging by pipetting, cover the egg chambers with a 20 x 20 mm pre-cleaned glass cover slip, and seal the cover slip on glass slide by putting nail polish at the edge of the cover slip. 
	NOTE1: Pre-clean the glass slide and cover slip with water or 70% ethanol. 
	NOTE2: Apply petroleum jelly between the glass slide and the cover slip to avoid destroying the egg chambers by overcompression.</li>
	<li>Image the egg chambers using fluorescence or confocal microscope, using a 20x, NA 0.8 Plan-Apochromat objective, with excitation light wavelength 405 nm for nuclear staining (detect emission ~461 nm), 561 nm for RFP (ongoing or recent caspase activity) signal (detect emission ~590 nm), and 488 nm for GFP (past caspase activity) signal (detect emission ~518nm). 
	Note: See our published protocols for microscopy20,64.</li>
</ol>

The authors have nothing to disclose.

The CaspaseTracker dual biosensor system is a novel and unique tool that allows detection of recent or ongoing caspase activity, and tracking of cells that have reversed cell death process and survive after experiencing caspase activity in vivo. While caspase activity has been traditionally assumed as a hallmark of apoptosis, growing studies reveal that non-apoptotic caspase activity plays potential roles in diverse normal cell functions, such as regulation of neuronal activity79,80, learning and memory81,82,83,84, suppression of necroptotic cell death85,86, spermatid individualization87,88, microRNA processing89, cell proliferation90, and cell fate patterning91. In addition to apoptosis and anastasis, the CaspaseTracker biosensor system can therefore detect non-apoptotic caspase activity, which is present in the brain and optic lobes, cardia, gut, Malpighian tubules, trachea, muscles, and other tissues of Drosophila19,46,64. This biosensor signal could also represent the current or past anastatic activity during embryo development or normal homeostasis in these tissues. To study anastasis after transient cell death-inducing environmental stress in live animals, it is critical to choose tissues with cells that exhibit no caspase biosensor activity under normal physiological conditions, but that can be induced to undergo caspase activation by transient cell death induction. Egg chambers are ideal for this purpose, because they typically have no caspase activity from germarium to stage 10 during oogenesis58,59,92.
Exposing female Drosophila to transient environmental stresses, such as protein starvation and cold shock, can efficiently trigger caspase activation and cell death in egg chambers19,57,58,78. Critical steps within the protocol include avoiding prolonged cell death induction to flies. The optimized conditions of protein starvation (8% sucrose in 1 % agar for 3-days) and cold shock (1 h&#160;at -7 oC) to female flies can trigger caspase-activated cell death process in egg chambers, and allow them to recover after the stressed flies are returned to normal condition19. Prolonged treatment with a cell death stimulus can trigger more egg chambers to undergo the cell death process, but the recovery rate is also reduced, presumably because the dying egg chambers experience massive damage beyond repair.
An additional critical step in this protocol is to reduce the CaspaseTracker background signal in egg chambers by crossing, raising and maintaining the CaspaseTracker flies at a lower temperature, such as 18 oC. While the majority of egg chambers from optimally reared flies do not display caspase activity in the germarium through stage 10 during oogenesis58, about 1% of egg chambers could exhibit caspase biosensor activity without cell death induction19. This may reflect the normal attrition rate due to innate errors or may be triggered unintentionally during oogenesis by standard laboratory conditions. As Gal4 displays less activity in flies at low temperature93, raising flies at 18 oC, rather than at room temperature, can reduce the endogenous signal that activates the CaspaseTracker system. Alternatively, switching flies to a higher temperature, such as 29 oC, can increase the sensitivity of CaspaseTracker system, due to increase in the Gal4 activity93, and potentially other endogenous temperature-dependent enzymatic activities.
It is important to distinguish the CaspaseTracker-positive cells that undergo cell death process or anastasis from cells that exhibit non-apoptotic caspase activity. Apoptotic cells express RFP (transient marker for on-going or recent caspase activity), and often GFP (permanent maker for past caspase activity) in treatment of apoptosis induction, as these dying cells have ongoing caspase activity that cleave-activated Gal4, which then activates the transient (Gal4 activity-dependent RFP) and permanent (Gal4 triggered FLPase-FRT mediated GFP) reporters of the G-Trace system. Apoptosis of these cells can be confirmed by morphological and biochemical hallmarks such us nuclear condensation detected by staining with nuclear dyes14,19,58, and caspase cleavage detected by immunostaining19,94. Cells that have already undergone anastasis display permanent GFP expression due to the FLPase-mediated recombination event of G-Trace system. These cells express no RFP as they have no on-going caspase activity, nor other hallmarks of apoptosis19. These cells also display normal nuclear morphology. Cells do have on-going non-apoptotic caspase activity often display both RFP and GFP expression, and also have a normal nuclear morphology19.
It can be difficult to distinguish cells that experienced anastasis from those cells that had past non-apoptotic caspase activity, because both display only GFP and have no hallmark of cell death. Therefore, careful control experiments must be included19. For examples, to study anastasis in egg chambers, it is essential to examine GFP expression in both stressed-recovered flies and non-stressed flies (negative control). Recovered flies should show more GFP-expressing cells than unstressed flies, if anastasis has occurred after caspase activation. It is also important to distinguish CaspaseTracker fluorescent signals from nonspecific auto-fluorescence, as observed in cuticle, pigments, and fat bodies19. We generated negative control biosensor flies by mutating the caspase cleavage site of the biosensor (DQVD) to a non-cleavable sequence (DQVA) and thereby rendering the control biosensor non-responsive to caspase activity19. The signal detected in the caspase sensitive (DQVD) but not in the negative control (DQVA) biosensor flies is the signal of interest, triggered by caspase activity, rather than auto-fluorescence.
Our current Drosophila dual CaspaseTracker biosensor can identify cells with &#34;recent&#34; caspase activity by expression of RFP, and cells with &#34;past&#34; caspase activity by expression of GFP19. We note that RFP is not a &#34;real-time&#34; caspase activity indicator, because it takes a few hours of reaction time for Gal4 to drive the expression of RFP in response to caspase activity. To add a &#34;real time&#34; function, the Drosophila CaspaseTracker biosensor can be combined with the recently developed iCasper biosensor52, a &#34;real-time&#34; and &#34;dark to bright&#34; in vivo biosensor that only shows its far-red signal when it is cleaved by caspases.
Apart from apoptosis, CaspaseTracker could potentially also be activated during alternative types of cell death as cells can switch cell death pathways during their course that directly or indirectly involves caspase activation, such as autophagy after starvation95,96, necrosis by the cold shock-induced plasma membrane rupture19,55,56,78, and staurosporine-induced necroptosis97,98. Our present and previous studies demonstrated that the CaspaseTracker biosensors can label cells recovered from these cell death inductions in vitro or in vivo17,18,19,20,21. As these cell death inductions alone could simultaneously activate multiple cell death pathways in the same cells, the recovery of these dying cells suggests that anastasis is a general cell recovery phenomenon that can reverse different cell death processes including apoptosis, autophagy, necrosis and necroptosis, individually or simultaneously.
The in vivo CaspaseTracker biosensor will facilitate pursuit of the yet unknown functions, mechanisms, and therapeutic implications of anastasis (Figure 4)21. To reveal the molecular signature of anastasis, we performed time-course whole-genome gene expression microarray studies to analyze mouse primary liver cells during reversal of ethanol-induced apoptosis, and interestingly, found striking changes in transcription of genes involved in multiple pathways including pro-survival, anti-oxidation, DNA damage response, histone modification, angiogenesis, cell migration, and transformation18,21. This finding is supported by our validation study of the recovery of human liver cancer HepG2 cells from apoptosis18,21, and also a recent independent RNA-sequencing study of HeLa cells in recovery of apoptosis99. Interestingly, up-regulation of some pro-survival factors found in anastatic cells are also observed in heart failure reversal, tumor progression, and cancer recurrence100,101,102, suggesting the potential participation of anastasis. To study the physiological, pathological and therapeutic potentials of anastasis, it is important to identify the anastatic cells and track their fate in small animals, as this will enable mechanistic and therapeutic studies. Our Drosophila and mammalian CaspaseTracker biosensors will be useful tools for testing the potential contributions of anastasis in normal development, homeostasis, tissue recovery, tumor evolution, cancer recurrence, and metastasis. Finding from these studies will increase our understanding of the natural role of anastasis, and offer potential to identify revolutionary new therapeutic approaches for intractable diseases by mediating cell death and survival through controlling anastasis.

Programmed cell death, such as apoptosis, plays an essential role in embryonic development and normal homeostasis by eliminating unwanted, injured, or dangerous cells in multicellular organisms1,2,3. The loss of balance between cell death and survival can lead to fatal consequences such as cancer, heart failure, autoimmunity, and degeneration4,5,6,7,8. Activation of executioner caspases has traditionally been considered as the &#8220;point of no return&#8221; in apoptosis9,10,11, as it triggers rapid and massive cellular demolition12,13,14,15,16. Challenging this general dogma, we demonstrated that cultured dying primary cells and cancer cells can recover not only after caspase activation, but also following important cell death hallmarks including plasma membrane blebbing, cell shrinkage, mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, nuclear and chromatin condensation, DNA damage, nuclear fragmentation, cell surface exposure of phosphatidylserine (PS), and formation of apoptotic bodies17,18,19,20,21. We propose that anastasis is an intrinsic cell recovery phenomenon, as dying cells can recover after removal of cell death stimuli17,18,19,20,21. We coined the term &#8220;Anastasis&#8221; (&#913;&#957;&#945;&#963;&#964;&#940;&#963;&#951;&#962;)18, which means &#8220;rising to life&#8221; in Greek, to describe this unexpected cell recovery phenomenon. Our observation of anastasis is further supported by recent independent studies that also reveal recovery of cells after phosphatidylserine externalization22,23,24, limited mitochondrial outer membrane permeabilization25,&#160; activation of mixed lineage kinase-like (MLKL), and cell shrinkage26.
Characterizing the mechanisms regulating anastasis will have paradigm-shifting physiological, pathological, and therapeutic implications. Anastasis could represent a previously unknown cytoprotective mechanism to rescue or preserve important postmitotic cells and tissues that are difficult to replace, and possibly account for heart failure reversal by ventricular unloading with left ventricular assist devices (LVADs)27,28, recovery of photoreceptor cells after transient exposure of excessive light29,30,31, or repair of neurons after brain injury32. If so promoting anastasis could enhance cell and tissue recovery. Conversely, anastasis could be an unexpected escape tactic used by cancer cells to survive cell-death-inducing therapy, causing cancer recurrence17,18. Therefore, suppressing anastasis in dying cancer cells during and after cancer treatment could be a novel therapeutic strategy to cure cancers by preventing their relapse.
During the process of anastasis, we have found that some recovered cells acquired permanent genetic alterations and underwent oncogenic transformation, likely due to DNA damage incurred during apoptosis18,20,21. Reversing the death process of DNA-damaged cells could be a mechanism of tumorigenesis, potentially underlying the observation that repeated tissue injury increases the risk of cancer in a variety of tissues, such as chronic thermal injury in the esophagus induced by the consumption of very hot beverages33,34,35, liver damage due to alcoholism36,37, tumor evolution after genotoxic cancer therapy38,39,40, and development of new cancers from normal tissues that arise during the intervals between cycles of anti-cancer therapy41,42,43,44. If true, targeting anastasis could prevent or arrest cancer development and progression. We have found that starvation-induced dying germ cells undergo anastasis in re-fed Drosophila19.&#160; If anastasis occurs in germ cells with DNA damage, it could be account for the observation that prolonged environmental stress promotes development of genetic diseases. For example, famines contribute to the development of transgenerational inheritable diseases such as diabetes and coronary heart diseases45. Therefore, understanding anastasis could lead to strategies for the prevention of developing inheritable diseases caused by this potential mechanism.
To harness the discovery of anastasis and direct it to develop innovative therapies, it is essential to study the cause and consequence of anastasis in live animals. However, it is technically challenging to identify and track anastatic cells in vivo, because the cells that recovered from cell death process appear morphologically indistinguishable from normal healthy cells, and there is no biomarker of anastasis identified yet17,18,21. To address these problems, we recently developed a new in vivo caspase biosensor designated &#8220;CaspaseTracker&#8221;19, to identify and track cells that survive apoptosis after caspase activation19,46, the hallmark of apoptosis10,14. Distinguishing it from the &#8220;real-time&#8221; caspase biosensors such as SCAT12,47, Apoliner48, CA-GFP49, ApoAlert18,50, C3AIs51 and iCasper52 that detect on-going caspase activity, the CaspaseTracker biosensor additionally features the ability to permanently label cells that express caspase activity even transiently. Therefore, the CaspaseTracker biosensor enables long term tracking of anastasis after reversal of caspase-mediated cell death process in vivo.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">CONSUMABLES AND REAGENTS</td>
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			<td style="width:225px;"></td>
			<td style="width:297px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Vectashield mounting medium</td>
			<td style="width:229px;">Vector Products</td>
			<td style="width:225px;">H-1000</td>
			<td>Antifade mounting medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield mounting medium (with DAPI)</td>
			<td style="width:229px;">Vector Products</td>
			<td style="width:225px;">H-1200</td>
			<td>Antifade mounting medium with DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td style="width:229px;">Ted Pella</td>
			<td style="width:225px;">#505 (110mm, #5)</td>
			<td>Dumont tweezer biology grade, stainless steel</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hanging Drop Slides</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:225px;">12-565B</td>
			<td>Glass slides</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td style="width:229px;">Molecular Probes</td>
			<td style="width:225px;">H1399</td>
			<td>DNA stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mitotracker Red CMXRos&#160;</td>
			<td style="width:229px;">Molecular Probes</td>
			<td style="width:225px;">M-7512</td>
			<td>Mitochondria stain</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Cleaved caspase-3 (Asp175) antibody</td>
			<td style="width:229px;">Cell Signaling Technology</td>
			<td style="width:225px;">#9661</td>
			<td>Stain for active fragment of caspase-3</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Bovine Serum Albumin (BAS)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:225px;">A8806</td>
			<td>Blocking agent for immunostaining</td>
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			<td height="21" style="height:21px;width:273px;">Phosphate Buffered Saline&#160;</td>
			<td style="width:229px;">VWR</td>
			<td style="width:225px;">114-056-101</td>
			<td>Medium for washing and immunostaining</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Triton&#8482; X-100</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:225px;">T8787</td>
			<td>Detergent for cell permeabilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name</td>
			<td style="width:229px;">Company</td>
			<td style="width:225px;">Catalog Number</td>
			<td>Comments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EQUIPMENT</td>
			<td style="width:229px;"></td>
			<td style="width:225px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM780 confocal microscope</td>
			<td style="width:229px;">Carl Zeiss</td>
			<td style="width:225px;">N/A</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Carl Zeiss Stereomicroscope Stemi 2000&#160;</td>
			<td style="width:229px;">Carl Zeiss</td>
			<td style="width:225px;">N/A</td>
			<td>Drosophila dissection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">AmScope Fiber Optic Dual Gooseneck Microscope Illuminator, 150W</td>
			<td style="width:229px;">AmScope</td>
			<td style="width:225px;">WBM99316&#160;</td>
			<td>Light source</td>
		</tr>
	</tbody>
</table>

detecting anastasis in vivo by caspasetracker biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Watch this Scientific Journal Video about Detecting Anastasis In Vivo by CaspaseTracker Biosensor at JoVE.com

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

detecting-anastasis-in-vivo-by-caspasetracker-biosensor

Nanyang Technological University

Research

JoVE Journal

Medicine

9.2K Views.  Johns Hopkins University School of Medicine. Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Video: Detecting Anastasis In Vivo by CaspaseTracker Biosensor

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

Preparation of Naringenin Solution for In Vivo Application

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The complete solubilization of the compound is necessary for its safe use and relatively stable results. Here, a protocol for preparing naringenin solution and its intraperitoneal administration in a high-fat diet and streptozotocin (STZ)-induced diabetic model is demonstrated as an example. A small amount of naringenin (3.52-6.69 mg) was used to test its solubilization in solvents, including ethanol, dimethylsulfoxide (DMSO), and DMSO plus Tween 80 reconstituted in physiological saline (PS), respectively. Complete solubilization of the compound is determined by observing the color of the solution, the presence of precipitates after centrifugation (2000 x g for 30 s), or allowing the solution to stand for 2 h at room temperature (RT). After obtaining a stable compound/phytochemical solution, the final concentration/amount of the compound required for in vivo studies can be prepared in a solvent-only (no PS) stock solution, and then diluted/mixed with PS as desired. The antidiabetic osteoporotic effects of naringenin in mice (intraperitoneal administration at 20 mg/kg b.w., 2 mg/mL) were assessed by measuring blood glucose, bone mass (micro-CT), and bone resorption rate (TRAP staining and ELISA). Researchers looking for detailed organic/phytochemical solution preparations will benefit from this technique.

Preparation of Naringenin Solution for In Vivo Application

Here, the protocol presents the preparation of naringenin solution for in vivo intraperitoneal administration. Naringenin is fully dissolved in a mixture of dimethylsulfoxide, Tween 80, and saline. The antidiabetic osteoporotic effects of naringenin were assessed by blood glucose testing, tartrate-resistant acid phosphatase staining, and enzyme-linked immunosorbent assay.

The preparation of a compound (phytochemical) solution is an overlooked but critical step prior to its application in studies such as drug screening. The ...