This work was supported by grants from the University of Buenos Aires (UBACyT 2018-2020 20020170100082BA), the National Council for Scientific Research and Technology (CONICET) (PIP 2021-2023 GI&#8722; 11220200101549CO; and PUE 22920170100033) and the National Agency for Scientific and Technological Promotion (PICT 2019-01664).

1. Cell preparation
<ol>
	<li>Soak 12 mm round coverslips in absolute ethanol, and place them vertically in the wells of a 24-well plate.</li>
	<li>Remove the cover, and expose the multi-well plate to ultraviolet radiation for 15 min.</li>
	<li>Position the coverslips horizontally, and wash them with Dulbecco&#39;s Modified Eagle Medium (DMEM).</li>
	<li>Seed a low passage number of pancreatic cells. The amount should be adjusted to obtain 50%-75% confluency on the day of fixation16. 
	NOTE: It is recommended to seed 2.5 &#215; 104 PANC-1 or 4 &#215; 104 AR42J cells per well to fixate the cells after 3 days.</li>
	<li>Culture the cells in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin in an incubator at 37 &#176;C under a humidified atmosphere with 5% carbon dioxide (CO2). 
	&#8203;NOTE: For PANC-1 cells, it is recommended to incubate the cells for 2 days between cell seeding and the following steps. After this time, the cells can be transfected, treated, or fixated. This protocol exemplifies the treatment with gemcitabine in non-transfected PANC-1 cells and the differentiation and PP242 treatment for non-transfected AR42J cells.</li>
</ol>
2. Treating the cells
<ol>
	<li>Gemcitabine treatment for PANC-1 cells
	<ol>
		<li>Prepare a solution of 1 &#181;g/&#181;L gemcitabine in DMEM 2 days after seeding. Treat each well with 2.6 &#181;L of the 1 &#181;g/&#181;L gemcitabine solution to achieve a final dilution of 20 &#181;M.</li>
		<li>Incubate the cells for 24 h in the incubator.</li>
	</ol>
	</li>
	<li>AR42J differentiation and PP242 treatment
	<ol>
		<li>Prepare a solution of 4 &#181;g/mL dexamethasone in DMEM.</li>
		<li>Treat each well with 4.9 &#181;L of 4 &#181;g/mL dexamethasone solution to obtain a final dilution of 100 nM.</li>
		<li>Incubate the cells for 48 h in the incubator.</li>
		<li>Remove the medium, and treat each well with 0.5 &#181;L of 1 mM PP242 to obtain a final dilution of 1 &#181;M.</li>
		<li>Incubate the cells for 2 h in the incubator.</li>
	</ol>
	</li>
</ol>
3. Fixing and permeabilizing the cells
<ol>
	<li>Prepare a 24-well plate with cold methanol and a 6-well plate with cold phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4). Maintain them on ice.</li>
	<li>Take each coverslip with tweezers, wash it twice in PBS, and incubate for 6 min in methanol.</li>
</ol>
4. Blocking the cells
<ol>
	<li>Wash each coverslip twice in PBS, and incubate for 1 h in 10% fetal bovine serum in PBS (blocking solution). 
	&#8203;NOTE: In this step, the protocol might be paused. The coverslips can be stored overnight in the fridge in the blocking solution, and the protocol can be continued the following day.</li>
</ol>
5. Incubating the coverslips with the primary antibody
<ol>
	<li>Prepare a 1:1,000 solution of anti-LC3 in the blocking solution, and maintain it on ice.</li>
	<li>Place a piece of laboratory sealing film over the multi-well lid.</li>
	<li>Place one drop (25 &#181;L) per coverslip of anti-LC3 solution over the sealing film.</li>
	<li>Take each coverslip with tweezers, and place it over the primary antibody drop, taking care that the cell side is in contact with the solution.</li>
	<li>Prepare a humid chamber by placing a humid piece of paper into a flat-bottom plastic box.</li>
	<li>Place the multi-well plate into the humidity chamber, cover it with foil, and incubate overnight in the fridge.</li>
</ol>
6. Incubating the coverslips with the secondary antibody
<ol>
	<li>Remove the multi-well plate from the humidity chamber, and place the coverslips back in the multi-well plate.</li>
	<li>Perform three washes with PBS.</li>
	<li>Prepare a solution of fluorescently labeled anti-rabbit with a dilution of 1:800 in the blocking solution, and maintain it on ice protected from light.</li>
	<li>Place a sealing film piece over the multi-well lid.</li>
	<li>Place a drop (25 &#181;L) per coverslip of anti-rabbit solution over the sealing film.</li>
	<li>Take each coverslip with tweezers, and place it over the primary antibody drop, taking care that the cell side is in contact with the solution.</li>
	<li>Incubate the multi-well plate in the humidity chamber for 2 h at room temperature (RT) protected from light.</li>
</ol>
7. Staining the cells with 4&#8242; ,6-diamidino-2-phenylindole (DAPI)
<ol>
	<li>Remove the multi-well plate from the humidity chamber, and place the coverslips back in the multi-well plate.</li>
	<li>Perform three washes with PBS.</li>
	<li>Prepare a 300 nM solution of DAPI in PBS (protected from light).</li>
	<li>Incubate each coverslip with the DAPI solution for 10 min.</li>
	<li>Perform three washes with PBS. Maintain the multi-well plate protected from light.</li>
</ol>
8. Montage
<ol>
	<li>Prepare two beakers with water and a piece of paper.</li>
	<li>Place one drop (10 &#181;L) per coverslip of a polyvinyl alcohol-Bis(trimethylaluminum)-1,4-diazabicyclo[2.2.2]octane adduct (PVA-DABCO) solution on a slide. 
	NOTE: PVA-DABCO is prepared by combining 0.25 M DABCO, 10% W/V PVA, 20% glycerol, and 50% Tris HCl (1.5 M, pH 8.8) in ultrapure water.</li>
	<li>Take each coverslip with tweezers, wash it in each water beaker, dry it off in the paper, and place it over the PVA-DABCO drop (with the cells in contact with the solution).</li>
	<li>Let it dry overnight, protected from light.</li>
</ol>
9. Confocal microscopy viewing and image capture
<ol>
	<li>Visualize the coverslips in an inverted confocal microscope using an objective of around 63x17.</li>
	<li>Capture representative images of the labeled cells.</li>
</ol>
10. Quantifying the LC3 dots
<ol>
	<li>Drag and drop each image file containing the captured channels, such as &#34;.czi&#34;, into the ImageJ (FIJI) screen to open. Click on Ok in the dialog box, and close the Console window.</li>
	<li>From the Image tab, select Color &#62; Split Channels.</li>
	<li>Close the images corresponding to the channels other than the LC3 image.</li>
	<li>From the Image tab, select Adjust &#62; Color Balance</li>
	<li>Move the Maximum slider to the left until the image is saturated to visualize the cell contours.</li>
	<li>Draw the cell outline with the Freehand Selection tool.</li>
	<li>Click on the Reset button to reset the color adjustment.</li>
	<li>From the Edit tab, select Cut to cut the selected item.</li>
	<li>Close the image without saving it.</li>
	<li>From the Edit tab, select Paste.</li>
	<li>In the Analyze menu, choose the tool 3D Objects Counter.</li>
	<li>Set the threshold. In the example provided in this study, the threshold is set at 2,000.</li>
	<li>Set the size filter. In this study, it is set between 50 and 500.</li>
	<li>Be sure that the boxes Objects and Summary are marked.</li>
	<li>Click on Ok. The number of dots will be described as Objects Detected in the Summary.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65005/65005fig01.jpg" /> 
Figure 1: Schematic diagram of the LC3 immunofluorescence protocol. Schematic diagram that represents the general protocol provided for LC3 immunofluorescence. Figure created with BioRender.com. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65005/65005fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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No conflicts of interest were declared.

The method described in this protocol allows for visualizing the endogenous LC3 distribution in the cell and quantifying the autophagic levels under different conditions. Another similar method used to analyze the LC3 distribution and determine autophagy activation involves fluorescence-labeled LC3 transfection (such as RFP-LC3)19. RFP-LC3 transfection has the advantages of not needing fixation (which allows for applying this method in live cell imaging20), being cheaper, and not depending on LC3 antibody reactivity. On the other hand, the immunofluorescence of LC3 has the advantage of providing an image of the endogenous LC3, thus avoiding possible issues related to LC3 overexpression, such as the formation of protein aggregates that are independent of autophagy21. Moreover, this method does not depend on the ease of transfecting the cells, meaning it is applicable to diverse cell lines. However, depending on the LC3 antibody that is used and its reactivity, there are some cellular lines in which it might not work. Some antibodies may work well for certain species but not for others, even when they are theoretically compatible with different species. In the case of the antibody used in this protocol (LC3B&#160;D11), we have found that it works perfectly for human cells (PANC-1, HEK293T, HeLa) and rat cells (AR42J). However, it does not work for mouse cells (MEF cells), as we observed nonspecific nuclear staining. It is worth noting that the quantification of LC3 spots, whether endogenous or overexpressed, has limitations in distinguishing between changes in autophagy activation, which may indicate the increased production of LC3-II, and changes in LC3-II degradation, which may indicate an autophagic flux state. Additional methods can be used to comprehensively assess autophagy activity. For instance, the use of RFP-GFP-LC3 expression can provide an accurate assessment by distinguishing between LC3-II inside or outside of the lysosome22,23.
As shown in Figure 4, there are some critical steps in the protocol that must not be modified, given that their modification can lead to suboptimal outcomes. First, it is important to set the correct number of cells to be seeded. When not enough cells are seeded, they tend not to resist transfection or treatments and remain rounded. On the contrary, when cells are seeded in excess, they tend to grow on top of the neighboring cells, making it challenging to focus on individual cells and distinguish between their LC3 dots. Notably, in situations where cells are in close proximity but not overlapping&#160;as depicted in Figure 4A, it may be helpful to use immunostaining with specific membrane markers, such as EGFR, to distinguish between the positive markers belonging to each cell24. However, it is important to note that certain markers like E-cadherin and EpCAM are not suitable for this purpose in PANC-1 cells due to their reduced membrane expression, which results from the typical epithelial-mesenchymal transition process associated with this cell type25,26,27. Secondly, when working specifically with PANC-1 cells, it is essential to wait at least 1 day between the seeding and the subsequent steps of the experiment. Conversely, when one does not wait for the right time, the cells can be rounded, making it difficult to interpret the results. Thirdly, fixation is a critical step in this protocol. As we have shown, the method only works with an adequate methanol fixation. Paraformaldehyde fixation does not work correctly for LC3 immunolabelling, while the methanol fixation time should not be modified, given that shorter times might lead to an incomplete fixation. We tested methanol fixation times up to 1 h and did not observe differences in LC3 labeling. Nevertheless, we advise against the use of prolonged fixation times, as methanol fixation can lead to the loss of soluble cellular proteins and free fluorescent molecules28. Therefore, it is recommended to adhere to the standard fixation time to ensure precise and dependable results.
Some modifications might be accepted in the described protocol. For example, a 12-well plate can be used instead of the 24-well plate, as well as growing the cells over 15 mm round coverslips instead of 12 mm coverslips. In this case, it should be considered that the number of cells to be seeded, as well as the volumes of reagents used, will be greater than those described in this protocol. In this case, 4 &#215; 104 PANC-1 and 6.5 &#215; 104 AR42J should be seeded. Additionally, the used blocking solution can be replaced with others, like 1% BSA in PBS, and this would lead to similar results. In the same way, the blocking time could be increased up to 24 h without significantly altering the results. The antibody incubation times and concentration might be adjusted and can change, for example, if another LC3 antibody is used. Although PVA-DABCO solution is prepared in this study, commercial montage solutions can also be used. On the other hand, the quantification method can be modified. For example, applying some filters or masks to the images is possible, and an alternative tool can be used for dot quantification.
In this work, we directed the application of the immunofluorescence of LC3 to study the behavior of pancreatic cancer cells. In these cells, autophagy is basally activated and might be modulated as a response to diverse stressful situations, such as chemotherapy, hypoxia, or nutrient deficiency11,12. The determination of autophagy in these cells may be applicable to studying the cellular response to chemotherapy, radiotherapy, or other treatments that modulate autophagy. As shown in Figure 3, the presented method can be applied in physiological models.&#160;Although, in this work, we focused on the quantification of autophagic levels, the immunofluorescence of LC3 might also allow the evaluation of the colocalization between LC3 and diverse proteins. It can serve, for example, as a mechanistic approach to mark proteins at different points in the autophagic process and evaluate whether the colocalization with LC3 is affected by some treatments or by the downregulation of some proteins. In this way, it could be determined, for example, whether some autophagy inhibitor interrupts the autophagic flux before or after LC3 conjugation. Finally, the method can also be adapted to tissue samples to determine autophagy activation in animal models or human biopsy samples.

Macroautophagy (commonly referred to as autophagy) is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles1,2. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis3. During autophagy, a double membrane vesicle is formed: the autophagosome. The autophagosome contains the cargo molecules and drives them to the lysosome for degradation1,4. 
Autophagosomes are decorated by the autophagic protein microtubule-associated protein light chain 3 (LC3)5. When autophagy is not induced, LC3 is diffused in the cytoplasm and nucleus in the LC3-I conformation. On the other hand, when autophagy is induced, LC3 is conjugated with a phosphatidylethanolamine in the membrane of the autophagic structures6. This new LC3 conformation is known as LC3-II1. The LC3 conformation shift causes changes in its cellular localization and its dodecyl sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) migration, which can be detected by techniques such as immunofluorescence and western blot5,7. In this way, LC3 conjugation is a key event in the autophagic process that can be used to measure autophagic activity.
The pancreatic acinar cell is a highly differentiated cell that, under healthy conditions, has a low rate of autophagy. However, in different physiological conditions or under pharmacological stimulation, they can activate autophagy. Therefore, the determination of autophagic levels in this cell line is useful for studying the potential direct or indirect effects of different pharmacological or biological agents on autophagy8,9.
Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer, given its late diagnosis and its high chemotherapy resistance10. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy-related proteins11. Pancreatic cancer cells may adjust their autophagy levels in response to unfavorable conditions like hypoxia, nutrient deprivation, or chemotherapy-induced damage11. Hence, analyzing the autophagy levels in pancreatic cancer cells can help understand how they adapt to varying environments and evaluate the effectiveness of autophagy-modulating treatments.
This study shows a method to perform LC3 immunofluorescence in two distinct pancreatic cellular models. The first model, PANC-1 cells, served as a model for pancreatic ductal adenocarcinoma. These cells were treated with gemcitabine, a chemotherapy agent that has previously been shown to induce autophagy, specifically in pancreatic cancer cells carrying the oncogenic Kirsten rat sarcoma virus gene (KRAS)12,13. The second model, AR42J cells, served as a more physiological model of exocrine pancreatic cells. These cells were differentiated with dexamethasone to become more similar to acinar pancreatic cells14. In these cells, autophagy was pharmacologically induced through the use of PP242, which is a potent mTOR inhibitor15. In this study, we demonstrate the applicability of the protocol described with two different pancreatic models and its ability to discriminate between states of low and high autophagy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Phosphate-Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm round coverslips</td>
			<td>HDA</td>
			<td>CBR_OBJ_6467</td>
			<td></td>
		</tr>
		<tr>
			<td>24 Well- Cell Culture Plate</td>
			<td>Sorfa</td>
			<td>220300</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Biopack</td>
			<td>2207.10.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Donkey anti-rabbit IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>R37119</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole, dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma Aldrich</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEN</td>
			<td>Sartorius</td>
			<td>01-052-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>NATOCOR&#160;</td>
			<td>Lintc-634</td>
			<td></td>
		</tr>
		<tr>
			<td>Gemcitabina</td>
			<td>Eli Lilly</td>
			<td>VL7502</td>
			<td></td>
		</tr>
		<tr>
			<td>LC3B (D11) XP Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>3868S</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Anedra</td>
			<td>6197</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm &#34;M&#34; (Laboratory Sealing Film)</td>
			<td>Bemis/Curwood</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strep Solution</td>
			<td>Sartorius</td>
			<td>03-031-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>PP242</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-301606</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Gibco</td>
			<td>11570626</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating autophagy levels in two different pancreatic cell models using lc3 immunofluorescence

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins.
Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the &#34;3D Objects Counter&#34; tool.
The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.

This protocol performs immunofluorescence of LC3 in pancreatic cell lines to determine the autophagy levels in different conditions. The outcome of this experiment was the obtention of cellular images from the red and blue channels, corresponding to LC3 and DAPI. The LC3 images indicate the cellular distribution of this protein, whereas the DAPI shows the nuclear localization. Figure 2A shows a representative image of the immunofluorescence of LC3 and its merge with DAPI staining in PANC-1 cells under basal or gemcitabine treatment conditions. A set of images of LC3 staining was analyzed using the tool 3D Objects Counter in FIJI. Using this software, the amount of LC3 dots per cell was quantified. The bar graph in Figure 2B shows the results of LC3 dot quantification in PANC-1 cells under basal versus gemcitabine treatment conditions. In this graph, the LC3 dots significantly increased under gemcitabine treatment, with the number of LC3 dots directly indicating the autophagic activity. We also previously demonstrated that gemcitabine triggers autophagy in pancreatic cancer cells12. Overall, the method presented in this article allows the detection of the level of increase in autophagy activation induced by gemcitabine in these cells.
While this protocol focuses on using LC3 immunofluorescence to determine autophagic activity in pancreatic cancer cells, it could potentially be applied to other cell lines, including more physiologically relevant models. To test the method&#39;s efficacy in assessing physiological responses, the AR42J cell line was used. Although these cells are derived from a rat exocrine pancreas tumor, they can be differentiated into exocrine cells with glucocorticoid stimulation, thus making them a suitable pancreatic model8,14,18. The AR42J cells were differentiated with 100 nM dexamethasone treatment for 48 h, followed by treatment with the mTOR inhibitor PP242 to induce autophagy15. The obtained results are presented in Figure 3, which shows a significant increase in the number of LC3 dots per cell under the PP242 treatment.
Thus far, we have demonstrated that the presented method is effective for assessing autophagic activity in both cancer cells and a more physiological model. However, it is important to note that minor deviations from the presented protocol could result in uninterpretable results.
Figure 4A shows a representative image from a suboptimal experiment in which too many cells were seeded on the coverslips, and excessive confluence was obtained. This kind of experiment might be uninterpretable for diverse reasons. Firstly, the cellular types mentioned in this work are derived from the exocrine pancreas, where the cells are grouped in acini. Under an excessive confluence, these cells tend to pile up and grow on top of each other (such as cell 1 and cell 2 in Figure 4A, which are above cell 3 and cell 4). This phenomenon makes it practically impossible to know which cell the LC3 dots belong to, thus making it very difficult to estimate the number of dots per cell. On the other hand, cells at high confluency tend to be stressed, which triggers autophagy. As a result, the differences in autophagic levels between the control and treated cells might decrease due to an increase in the background autophagic activity.
In Figure 4B, a representative image from another kind of suboptimal experiment is shown in which the cells were fixed with paraformaldehyde instead of methanol. While this fixation method is generally effective for preserving a variety of proteins, it is not suitable for LC3, as the resulting image does not accurately reflect its true distribution. This technical mistake might make it impossible to find differences between low and high autophagic levels.
Generally, cell lines can be treated, transfected, or fixed 1 day after seeding. Nevertheless, it is crucial to mention that, in the case of PANC-1 cells, it is necessary to wait for 2 days after seeding to ensure the complete adherence of the cells to the glass coverslips before proceeding with the subsequent steps of the experiment. Figure 4C shows a representative image from an experiment in which the cells were treated with gemcitabine just 1 day after seeding. From the figure, it can be observed that the cells in this experiment had a round shape. This morphology decreased the relation between the cytoplasm and nucleus, making it difficult to understand the intracellular distribution of LC3 and to discriminate between low and high autophagic levels. It is important to note that AR42J does not have this problem, and they are ready to be treated or fixed on the day following the seeding.
Another suboptimal outcome could be obtained when the time of methanol fixation is varied. Shorter times of fixation might cause incomplete fixation, as represented in Figure 4D, where the cells were fixed for 3 min. Incomplete fixation can interfere with proper LC3 immunolabeling, leading to unclear images and suboptimal quantification.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65005/65005fig02.jpg" /> 
Figure 2: LC3 immunofluorescence in PANC-1 cells under basal or gemcitabine conditions and LC3 dot quantification. The PANC-1 cells were either treated with 20 &#181;M gemcitabine for 24 h or left untreated and then immunolabelled with anti-LC3. (A) Representative images of each condition are shown. Scale bar: 10 &#181;m. (B) The bar graph represents the means and standard errors of the means (SEM) of the LC3 dots per cell for each condition. N = 10 cells per condition from three independent experiments. *** p &#60; 0.001 by a Student&#39;s t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65005/65005fig02large.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/65005/65005fig03.jpg" /> 
Figure 3: LC3 immunofluorescence in AR42J cells under basal or PP242 conditions and LC3 dot quantification. The AR42J cells were differentiated with 100 nM dexamethasone for 48 h and then either treated with 1 &#181;M PP242 for 2 h or left untreated, followed by immunolabelling with anti-LC3.&#160;(A) Representative images of each condition are shown. Scale bar: 10 &#181;m. (B) The bar graph represents the means and standard errors of the means (SEM) of the LC3 dots per cell for each condition. N = 10 cells per condition from three independent experiments. ** p &#60; 0.01 by a Student&#39;s t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65005/65005fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65005/65005fig04.jpg" /> 
Figure 4: Suboptimal experiments. Representative images of LC3 immunofluorescence in suboptimal experiments are shown. Scale bar: 10 &#181;m. (A) Excess confluence: 7 &#215; 104 PANC-1 cells were seeded, treated with gemcitabine, and immunolabelled with anti-LC3. Four cells are marked to show that cell 1 and cell 2 are above cell 3 and cell 4. (B) PFA fixation: PANC-1 cells were treated with gemcitabine, fixated with PFA, and immunolabelled with anti-LC3. (C) Incomplete stretch: PANC-1 cells were treated with gemcitabine the day after seeding and immunolabelled with anti-LC3. (D) Incomplete fixation: PANC-1 cells were treated with gemcitabine, fixated with methanol for 3 min, and immunolabelled with anti-LC3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65005/65005fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence at JoVE.com

Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

The goal of this protocol is to determine autophagic levels in pancreatic cancer and pancreatic acinar cells through LC3 immunofluorescence and LC3 dot quantification.

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows ...

evaluating-autophagy-levels-two-different-pancreatic-cell-models

Nanyang Technological University

Research

JoVE Journal

Biology

1.4K Views.  Universidad de Buenos Aires, CONICET. The goal of this protocol is to determine autophagic levels in pancreatic cancer and pancreatic acinar cells through LC3 immunofluorescence and LC3 dot quantification.

Video: Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes are critical for the investigations of spindle assembly and chromosome segregation in male meiotic division. The mouse spermatocyte is an ideal model for mechanistic studies of meiosis, however, the effective methods for the analyses of spermatocytes are lacking. In this article, a practical and efficient method for the in vivo inhibition of kinesin-7 CENP-E in mouse spermatocytes is reported. A detailed procedure for testicular injection of a specific inhibitor GSK923295 through abdominal surgery in 3-week-old mice is presented. Furthermore, described here is a series of protocols for tissue collection and fixation, hematoxylin-eosin staining, immunofluorescence, flow cytometry and transmission electron microscopy. Here we present an in vivo inhibition model via abdominal surgery and testicular injection, that could be a powerful technique to study male meiosis. We also demonstrate that CENP-E inhibition results in chromosome misalignment and metaphase arrest in primary spermatocytes during meiosis I. Our in vivo inhibition method will facilitate mechanistic studies of meiosis, serve as a useful method for genetic modifications of male germ lines, and shed a light on future clinical applications.

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

This article reports an in vivo inhibition of CENP-E through abdominal surgery and testicular injection of GSK923295, a valuable model for male meiotic division. Using the immunofluorescence, flow cytometry and transmission electron microscopy assays, we show that CENP-E inhibition results in chromosome misalignment and genome instability in mouse spermatocytes.

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes ...