Name: Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells (Video) | JoVE Journal
Uploaded: 2012-10-11T13:00:00.000+00:00
Duration: 12 min 44 s
Description: Learn to visualize programmed cell death in mouse embryos and differentiating embryonic stem cells using LysoTracker dye staining. This protocol enables rapid detection of cell death patterns in developmental biology research.

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5 minutes

We thank members of the Mariani lab for help editing the protocol. This work was funded by a CIRM Postdoctoral Training Grant (JLF), a CIRM BRIDGES internship (TZTT), the Robert E. and May R. Wright Foundation (FVM), and the University of Southern California (FVM).

1. LysoTracker Staining of Mouse Embryos
 <ol>
 <li>Generate mouse embryos by placing young (5-6 week old) females into male stud cages. Monitor on following mornings for the appearance of a vaginal plug indicating that mating has occurred. If the female is pregnant, noon on that day is called 0.5 dpc (days post coitus). The procedure presented here is ideal for embryos 7-13 dpc.</li>
 <li>On the embryonic day of interest, euthanize the female according to approved protocols and remove the uterus. Remove the embryos from the decidua in a 10 cm Petri dish with Hanks BSS (without phenol red). Remove the extra-embryonic membranes and do at least one rinse with Hanks BSS to remove extraneous tissues and blood.</li>
 <li>Prepare LysoTracker Embryo Staining Solution (5 μM LysoTracker in Hanks BSS). A convenient volume for 1-2 mouse litters is 5 ml. Gently mix to disburse the dye evenly.</li>
 <li>Add embryos to the LysoTracker Embryo Staining Solution in a microfuge tube or vial and incubate at 37 °C for 45 min.</li>
 <li>Rinse very gently in Hanks BSS four times, 5 min each wash.</li>
 <li>Fix in 4% Paraformaldehyde overnight.</li>
 <li>Rinse once in Hanks BSS, 10 min, to remove the fix.</li>
 <li>Dehydrate through a methanol series (50%, 75%, 80%, 100%, 5 min each step) to eliminate background staining. This is important for achieving good signal-to-noise during imaging.</li>
 <li>At this point you can store the embryo samples indefinitely in methanol at -20 °C, protected from light.</li>
 </ol>
 Note: A tissue sample can be taken at any step for DNA genotyping, however, it can be most convenient to take a sample just before storage so that up to this step all the samples can be batch-processed. Collect a sample of tissue from the tail, limb, or head from each embryo and put all into separate tubes. Rehydrate the tissue sample and prepare for genotyping.
 2. LysoTracker Staining of Differentiating ES Cultures
 <ol>
 <li>Prepare embryoid bodies via the Hanging Drop Method15 with a starting density of 500 cells per 20 μl drop.</li>
 <li>After 3 days, transfer the embryoid bodies to suspension culture on 10 cm non-adherent Petri dishes (i.e. those used for bacteriological work). Culture for 5 days, changing the media every 2 days.</li>
 <li>On day 7, transfer the embryoid bodies to gelatinized 8-well chamber slides (add 0.1% Gelatin to the chambers, wait 5 min, remove, and add media). Transfer 1-2 embryoid bodies per chamber.</li>
 <li>Culture for 10 days, changing media every other day. Be careful to place the pipette at the corner of the chamber slide so as to not perturb the embryoid body attaching to the glass surface. Also when adding fresh media also place the pipette at the corner of the chamber and release the media slowly.</li>
 <li>After 10 days of culture, to induce apoptosis as a positive control, treat some of the wells with 0.1 mM and 1.0 mM H2O2 (add the H2O2 to fresh media, mix, then add to the cells) for 60 min. Rinse twice with D-PBS and culture overnight in regular media.</li>
 <li>Gently aspirate existing media and add LysoTracker Cell staining solution (500 nM LysoTracker (final concentration) in media, 300 μl per chamber).</li>
 <li>Incubate at 37 °C for 15 min.</li>
 <li>Rinse twice with D-PBS 2 times, 5 min each.</li>
 <li>Fix in 4% paraformaldehyde for 15 min at RT.</li>
 <li>Rinse twice with D-PBS, 5 min.</li>
 </ol>
 3. Viewing LysoTracker Stained Embryos or ES Cultures
 <ol>
 <li>Prepare the cell samples for microscopy by aspirating the D-PBS; removing the chamber off the slide; air drying for about 5 min; and then adding an aqueous mounting medium (Vectashield with DAPI or similar). Place a foil barrier while drying to decrease bleaching of the fluorophore. Mount embryos in a deep-depression glass dish or small Petri dish in Vectashield or similar. </li>
 <li>Visualize with a dissecting or compound microscope outfitted with a rhodamine or Texas Red filter (LysoTracker RED DND-99, Excitation/Emission: 577/590 nm). The staining is typically quite bright so long exposures are usually not necessary.</li>
 </ol>
 4. Quantification of Results Using Imaging Techniques
 <ol>
 <li>Take digital photos of your samples under the same exposure conditions in manual mode.</li>
 <li>Open the images in Adobe Photoshop or a similar imaging program. To determine the amount of LysoTracker staining, select the red channel and threshold the image (converts the image to black-and-white) such that the red staining is now represented by white pixels. Select the white pixels and use the histogram function to obtain the total count.</li>
 <li>Use the blue channel to count the number of nuclei in the field. It can be useful to keep a record of the count by annotating the image.</li>
 <li>Record the values and then calculate the average staining level per cell. Repeat this analysis with the same threshold setting for all images you wish to sample.</li>
 </ol>
 5. Representative Results
 There is a normal pattern of PCD during embryonic development that can change when a growth factor gene, such as Sonic hedgehog (Shh), is removed16,17 . Since LysoTracker is a small molecule dye that is highly diffusible, the whole embryo can be visualized to assess areas of PCD even deep within the mesenchyme12,13. Figure 1 shows the LysoTracker staining pattern for normal and Shh-/- mutant mouse embryos at 10.5dpc. At this stage, PCD is greatly increased in Shh-/- mutant embryos, particularly in the limb and somite regions. Portions of the embryo can be imaged in whole-mount at higher magnification (1B) to assess the overall pattern. Dissected portions can also be sectioned with a vibrating microtome (1C) or a cryostat (D-F) to reveal PCD regions in greater detail.
 TUNEL analysis is best done on thin sections where penetration of the terminal transferase is less problematic. LysoTracker staining and a TUNEL assay can be done in the same tissue (Figure 1D-G). These results show that although there is not a strict 1:1 correlation between LysoTracker and TUNEL staining, the general regions of PCD overlap considerably. At a higher magnification, it is possible to see puncta that stain positive with the TUNEL assay but not with the LysoTracker dye, puncta that stain only with the LysoTracker dye, and puncta that have overlapping staining.
 When an EB is plated on a gelatin-coated surface, differentiating cells will migrate outward. After 10 days of culture, cells will have differentiated into a variety of cell types including neurons, muscle cells, and cardiomyocytes. The cultures shown in Figure 2 contain mixed populations of these cell types, however one could adapt our protocol to study PCD of a particular cell-type of interest. For example, the EB could be treated with Retinoic Acid to enhance the differentiation of neurons19 , and then the LysoTracker assay could be used to assess neuronal cell death under different conditions. In the normal culture of differentiated ES cells, there are a few cells that undergo PCD and these can be detected with LysoTracker and/or TUNEL assays (Figure 2A). PCD can be induced by a number of different cytotoxic agents such as hydrogen peroxide (H2O2) which induces oxidative damage and initiates the apoptotic pathway20,21 . A low dose of H2O2 increases both LysoTracker and TUNEL staining compared to no treatment, while a high dose results in cell shrinkage and widespread LysoTracker and TUNEL positivity. Similar to the results in the embryo, it is possible to see puncta that stain positive with the TUNEL assay but not with the LysoTracker dye, puncta that stain only with the LysoTracker dye, and puncta that have overlapping staining. The consequence of the application of a cytotoxic agent that induces apopotosis is compared to serum starvation conditions which induce autophagy (Figure 2D). Cells that have undergone a period of serum starvation (1 hour) exhibit a different pattern where LysoTracker and TUNEL staining do not highly correlate. LysoTracker staining is much increased compared to untreated cultures, however TUNEL positivity is at normal levels.
 Employing standard tools available in Adobe Photoshop, LysoTracker staining can be quantified with a simple thresholding method that allows one to calculate the average staining level for a population of cells. Figure 3A-B shows how a sample image looks during the quantification process. Starting with a baseline image with LysoTracker staining in the red channel and the DAPI-stained nuclei in the blue channel (panel 3A), the red channel of this image was thresholded to convert the image to black-and-white and the white pixels were then selected and counted (Figure 3A'). A thresholding level was chosen that represents the LysoTracker staining pattern. As with any quantification method that uses images, it is important to avoid overexposing your photos as this could potentially result in over-emphasizing differences. The number of the nuclei in the field can then be counted by looking at just the blue channel (Figure 3B) and marking the nuclei using a drawing tool such as a paintbrush while counting (Figure 3B'). One can then express the level of staining by calculating the average level of staining per cell (number of pixels/nuclei count). If one wishes to use this algorithm for high through-put screening, most screening packages can measure brightness in a particular channel and also have an object identifier function that will count nuclei in a field, however for most typical applications the manual method is quick and simple. To give an example, a sample region from Figures 2A-C was quantified and the results confirm that increasing concentrations of H2O2 results in a higher level of LysoTracker staining (Figure 3C).
 <img src="/files/ftp_upload/4254/4254fig1.jpg" alt="Embryo development comparison; LysoT and TUNEL staining, fluorescence microscopy analysis." data-altupdatedat="1787630408000" data-alt="Figure 1"> 
 Figure 1. LysoTracker staining in normal and Shh-/- embryos. A,A'. Normal and Shh-/- mouse embryos at 10.5 dpc, stained with LysoTracker Red, mounted in Vectashield and photographed under an epiflourescence dissecting microscope. Brackets indicate the location of the forelimbs. B,B'. Forelimb of normal and Shh-/- embryos imaged at higher magnification. Note the normal domains of cell death including the 'Posterior Necrotic Zone' (marked with an asterisk) and a region in the proximal mesenchyme of the limb bud (marked with an arrow). Shh null limbs exhibit stronger staining in the medial and distal portion. C,C'. Medial sections through the forelimb bud of both normal and null limb buds. Note the presence of staining in the proximal mesenchyme (arrow) and AER (arrowhead) of the normal limb bud and the strong staining within the dorsal and ventral and distal mesenchyme within the null limb bud. D-F. The same section (12 micron, frozen section) was imaged for LysoTracker and TUNEL staining (Roche 1684795). The section passes through the distal portion of the Shh null mesenchyme. The arrowhead points to the AER. Note how the overall pattern is very similar using both methods, however the staining does not overlap completely. G,G'. At a higher magnification of the AER and mesenchyme, one can see both the presence of puncta that are LysoTracker or TUNEL-positive only as well as puncta that have overlapping staining. 
 <img src="/files/ftp_upload/4254/4254fig2.jpg" alt="LysoTracker and TUNEL staining fluorescence microscopy; cell apoptosis analysis under H₂O₂ stress." data-altupdatedat="1787630408000" data-alt="Figure 2"> 
 Figure 2. LysoTracker staining in differentiated ES cells. Differentiating cells were plated on 8-chamber slides in the absence of LIF. To show how LysoTracker and TUNEL staining compare, the apoptosis pathway was induced with 2 different doses of H2O2, while the autophagy pathway was induced using 1 hour of serum starvation. These images were taken with a Zeiss LSM5 Confocal Microscope. A-A'': Untreated cultures show some normal apoptosis as indicated by LysoTracker and TUNEL staining. The pattern using both techniques mostly overlaps (white arrows), however, one can see a few puncta that are LysoTracker positive only (red arrows). B-B'', C-C": Treatment with increasing amounts of H2O2 stimulates apoptosis. The lower dose shows more LysoTracker and TUNEL positive puncta when compared to untreated cells. The higher dose induces in even more LysoTracker and TUNEL positive puncta. White arrows point to overlapping staining, while red arrows point to puncta that are positive for LysoTracker only. It is also possible to see puncta that are only positive for TUNEL staining (green arrows). D-D": Serum starvation can induce autophagy, which can stimulate an accumulation of material in lysosomes and autophagolysosomes. Note how many more LysoTracker puncta are visible compared to untreated cultures. TUNEL positive puncta are present (most of which have overlapping LysoTracker staining, white arrows), however, the number is not greatly increased compared to untreated conditions. In addition, under serum starvation conditions, LysoTracker-only positive puncta are highly prevalent (red arrows).
 <img src="/files/ftp_upload/4254/4254fig3.jpg" alt="LysoTracker fluorescence assay, nuclei staining, H₂O₂ response chart; microscopy, cellular analysis." data-altupdatedat="1787630408000" data-alt="Figure 3"> 
 Figure 3. Quantification method to assess the level of staining in a population of cells using Adobe Photoshop. A: A portion of Figure 2A was chosen and the LysoTracker (red channel) and DAPI staining (nuclei, blue channel) is shown in this panel. A': The red channel of Figure 3A was thresholded to convert it to a black-and-white image. The white pixels can then be selected and counted using the histogram function. B-B': The blue channel shows the nuclei and while counting them, the image can be annotated with a drawing function such as a paintbrush. C: This is an example quantification of a sample from panels 2A-C. The data is represented as pixels per nuclei. <a href="https://www.jove.com/files/ftp_upload/4254/4254fig3large.jpg" target="_blank">Click here to view larger figure</a>.

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No conflicts of interest declared.

Important hallmarks of apoptosis include the detection of DNA damage with the TUNEL assay, along with the detection of caspase activity (detected with mAbs or a binding molecule such as ZVAD-fmk), the observation of cellular changes, and the presentation of phosphatidylserine (PS) on the membrane surface (detected by Annexin V binding). There are some disadvantages with each of these assays. For example, the terminal transferase used in the TUNEL assay does not penetrate beyond a few cell layers. Also Annexin V can...

<table><tbody><tr><td>LysoTracker Red DND-99</td><td>Invitrogen</td><td>#L-7528</td><td></td></tr><tr><td>Hanks BSS</td><td>Invitrogen</td><td>14025-076</td><td></td></tr><tr><td>Paraformaldehyde</td><td>EMD</td><td>EM-PX0055-3</td><td></td></tr><tr><td>Vectashield</td><td>VECTOR</td><td>H-1200</td><td></td></tr><tr><td>DMEM</td><td>Cellgro</td><td>10-013-CV</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Cellgro</td><td>25-025-CI</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Cellgro</td><td>25-000-CI</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30071.02</td><td></td></tr><tr><td>Pen-Strep</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>b-Mercapt&amp;#8211;thanol, 50 mM </td><td>Invitrogen</td><td>21985-023</td><td></td></tr><tr><td>LabTek-II Chamber slides &lt;br/&gt; (8-well)</td><td>Nalge Nunc International</td><td>154534</td><td></td></tr><tr><td>0.1% Gelatin</td><td>Millipore</td><td>ES-006-B</td><td></td></tr><tr><td>Dulbecco&#039;s PBS (D-PBS) </td><td>Cellgro</td><td>21-031-CV</td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Solution Recipes&lt;/p&gt; &lt;p class=&quot;jove_step&quot;&gt;4% Paraformaldehyde&lt;/p&gt; &lt;p class=&quot;jove_content1&quot;&gt;For 100 ml:&lt;/p&gt; &lt;ol&gt; &lt;li&gt;Mix 4 g paraformaldehyde, 90 ml H&lt;sub&gt;2&lt;/sub&gt;O, and NaOH (a drop of 2N NaOH). The paraformaldehyde will not go into solution until you have added some NaOH to increase the pH.&lt;/li&gt; &lt;li&gt;Stir and heat at 60 &amp;deg;C until all the powder is in solution (~10-20 min). &lt;strong&gt;Do not overheat.&lt;/strong&gt;&lt;/li&gt; &lt;li&gt;Add ~10 ml 10x PBS to achieve a final volume of 100 ml.&lt;/li&gt; &lt;li&gt;Store at -20 &amp;deg;C in convenient (~10 ml or ~40 ml) aliquots.&lt;/li&gt; &lt;/ol&gt; &lt;p class=&quot;jove_content1&quot;&gt;&lt;strong&gt;WARNING: Paraformaldehyde in &#039;frill&#039; form (compressed small pellets) is less powdery and can therefore be measured outside of a hood. However you should still wear a protective dust mask (N95 at least) during handling.&lt;/strong&gt;&lt;/p&gt; &lt;p class=&quot;jove_step&quot;&gt;EB Culture Media&lt;/p&gt; &lt;p class=&quot;jove_content1&quot;&gt;For 500 ml EB Media:&lt;/p&gt; &lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td&gt;&lt;strong&gt;DMEM:&lt;/strong&gt;&lt;/td&gt; &lt;td&gt;&lt;strong&gt;404.5 ml&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;FBS:&lt;/td&gt; &lt;td&gt;75.0 ml&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;L-Glutamine:&lt;/td&gt; &lt;td&gt;5.0 ml&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Penicillin/Streptomycin:&lt;/td&gt; &lt;td&gt;5.0 ml&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Non-essential amino acids:&lt;/td&gt; &lt;td&gt;5.0 ml&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Sodium pyruvate:&lt;/td&gt; &lt;td&gt;5.0 ml&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;&amp;#946;-Mercapt&amp;#8211;thanol:&lt;/td&gt; &lt;td&gt;500 &amp;mu;l&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt; &lt;/table&gt;</td></tr></tbody></table>

use of lysotracker to detect programmed cell death in embryos and differentiating embryonic stem cells

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 ...

The aim of the following procedure is to demonstrate how to sustain the lysosomes of two types of samples, mouse embryos and differentiated stem cells with lyo tracker dye to detect patterns of cell death. To prepare embryo samples, embryos are first surgically isolated and placed into a dish to prepare differentiated cells. Embryonic stem cells are used to create embryo bodies.These embryo bodies are cultured in suspension and then grown as a monolayer on a chamber slide. The embryos all sell monolayer can be incubated in ot tracker red and are then rinsed and fixed samples can then be photographed to examine and quantify the staining results are obtained that show Liso Tracker. Staining is a simple procedure that can be used to assay program cell death in embryos and cultured cells.The main advantage of this technique over existing methods like tunnel analysis is that because OT tracker dye is a small molecule, thick tissue can be assayed quickly and easily. Though this method can provide insight into patterns of programmed cell death in mouse embryos and differentiated cultures, it can also be applied to other tissue types such as chicken embryos and differentiating adult stem cells. Pair young female mice with a stud male and on following mornings, check for a vaginal plug, which indicates the mating has occurred.Noon on the day that the vaginal plug appears is effectively 0.5 days. Post cotus on the embryonic day of interest, euthanize the female according to approved protocols and remove the uterus to a 10 centimeter Petri dish with Hank's BSS and rinse away any blood. Separate the embryos and remove them from the uterus.Remove the extra embryonic membranes and do at least one rinse with Hank's BSS to remove extraneous tissues and blood. Keep the dissected embryos on ice while cleaning. The others.Prepare five milliliters of Liso tracker embryo staining solution. This is a sufficient volume for one or two embryo collections. Gently mix the solution to disperse the dye, evenly the then transfer the embryos to the solution and incubate them at 37 degrees Celsius for 45 minutes.After the incubation, gently rinse the embryos in Hank's BSS four times for five minutes each wash. It is important to do the rinses gently so as to not damage the tissue. Remove the liquid, slowly paying close attention to where the delicate embryos are, and to always leave a portion of the liquid behind covering them.Then fix the embryos in 4%para formaldehyde overnight the next day. Rinse the embryos once in Hank's BSS for 10 minutes to remove the fixative. Now dehydrate the tissues through a methanol series to eliminate background staining.This step improves the signal to noise ratio during imaging prior to storage. If needed, collect a tissue sample from each embryo for genetic analysis. Later, rehydrate the tissue sample prior to genotyping.Separate the embryos into individual tubes with labels that match the tissue sample. Now it is possible to store the isolated embryos indefinitely in methanol of minus 20 degrees Celsius. Protected from light.Begin by preparing embryo bodies via the hanging drop method. With a starting density of 500 cells per 20 microliter drop after three days, transfer the embryo bodies to suspension cultures on 10 centimeter non-adherent Petri dishes. Culture them for five days, changing the media every two days after five days.Transfer the embryo bodies to gelatinized eight chamber slides, placing one or two embryo bodies in each chamber. Culture the embryo bodies for another 10 days. Changing the media every other day after 10 days, gently aspirate the existing media and add 300 microliters of Lyo tracker cell staining solution to each chamber.Be careful to manually pipette out the media placing the pipette tip in the corner of the chamber. This way, you won't accidentally remove the, still attaching every body from the glass surface. When adding back media again, place the pipette tip in the corner of the chamber and add the media back very slowly.Incubate the chambers at 37 degrees Celsius for 15 minutes. Then rinse them twice with DPBS for five minutes per rinse. Now fix the cells in 4%paraldehyde for 15 minutes at room temperature.Then remove the fixative with DPBS in two five minute rinses. To mount the embryos, place them in a deep depression glass slide or small Petri dish infector shield. Prepare the cell samples for microscopy by aspirating the DPBS.Remove the chamber from the slide and allow it to air dry for about five minutes. Cover with foil to protect the fluro. Then add an aqueous mounting medium such as vector shield with dappy and place a cover slip on top.Now the slide has been mounted and is ready for imaging. Visualize the samples with a dissecting or compound microscope outfitted with a rod, domine or Texas red filter. The staining is typically quite bright, so long exposures are usually not necessary in manual mode.Take digital photos of all the samples under the same exposure conditions. To quantify the images, open them in Adobe Photoshop or a similar imaging program. To determine the amount of lyo tracker staining, select the red channel.Note the pixel count for the entire image as revealed by the histogram function. Next threshold, the image such that the red staining is now represented by white pixels. This converts the image to black and white.Pick a threshold level and use this level for all images in the set. Select the white pixels using the color range tool and use the histogram function to obtain the total white pixel count. Use the blue channel to count the number of nuclei in the field.It can be useful to keep a record of the count by annotating the image, record the values and use them to calculate the average staining level per cell. Repeat this analysis with the same threshold setting for all the remaining images. To sample ly o tracker was used to stain sonic hedgehog mutant embryos.Since ly O tracker is a small, highly diffusible molecule areas deep within the meen of a whole embryo as stained at 10.5 days post cotus programmed cell death or PCG was greatly increased in sonic hedgehog null mutant embryos, particularly in the limb and so might regions. Limb buds were imaged in hole mounts at higher magnification to assess the overall pattern. Dissected portions were also sectioned with a vibrating microtone.The arrowhead points to increased staining in the sonic hedgehog null distal tip in sections obtained with a cryostat tunnel staining was used in conjunction with OT Tracker in these slices, although there is not a strict one-to-one correlation between lyo tracker and tunnel staining, the general regions of PCD overlap considerably under higher magnification. It is possible to see puncture that stained positive with the tunnel assay, but not the otra die puncture that stain only with the lyra die and puncture that have overlapping staining. After 10 days of embryo body culture cells differentiated into a variety of cell types, including neurons, muscle cells, and cardiomyocytes.In the normal culture of differentiated ES cells, there are a few cells that underwent PCD as detected with lyo tracker and tunnel assays. Next cells were treated with hydrogen peroxide to induce oxidative stress cell death. A low dose of hydrogen peroxide increased both lyo tracker and tunnel staining compared to no treatment.Similar to the results in the embryo, both stains labeled common and unique cells. A high dose of hydrogen peroxide resulted in cell shrinkage and more widespread OT tracker and tunnel positivity. For comparison, serum starvation was used to induce autophagy after one hour of serum starvation.OT tracker staining increased. However, tunnel positivity remained at normal levels. Thus, while reer is easy and useful, it is important to follow up with additional assays to determine if the staining represents apoptosis or autophagy after the samples were quantified.Using the simple Photoshop method demonstrated earlier, results confirmed that increasing concentrations of hydrogen peroxide resulted in a higher level of lys tracker staining Once mastered, this staining procedure can be done in less than two hours if it is performed properly Following this procedure. Other methods like NX in five or lc three immuno staining can be performed in order to confirm the type of cell death. Don't forget that currently Trecker green only works on live cells, so Reca red is your best option for samples that require fixation.

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

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