Name: evaluating autophagy levels in two different pancreatic cell models using lc3 immunofluorescence
Uploaded: 2023-04-28T13:40:00
Description: Watch this Scientific Journal Video about Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence at JoVE.com

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</...

<ol>
	<li>Grasso, D., Renna, F. J., Vaccaro, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+steps+in+mammalian+autophagosome+biogenesis.">Initial steps in mammalian autophagosome biogenesis.</a> Frontiers in Cell and Developmental Biology. 6, 146 (2018).</li><li>Galluzzi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+definitions+of+autophagy+and+related+processes.">Molecular definitions of autophagy and related processes.</a> EMBO Journal. 36 (13), 1811-1836 (2017).</li><li>Kitada, M., Koya, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+metabolic+disease+and+ageing.">Autophagy in metabolic disease and ageing.</a> Nature Reviews Endocrinology. 17 (11), 647-661 (2021).</li><li>Ktistakis, N. T., Tooze, S. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer.">Pancreatic cancer.</a> Lancet. 395 (10242), 2008-2020 (2020).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+function+of+autophagy+in+pancreatic+cancer.">Regulation and function of autophagy in pancreatic cancer.</a> Autophagy. 17 (11), 3275-3296 (2021).</li><li>Ropolo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+E2F1-EP300-VMP1+pathway+mediates+gemcitabine-induced+autophagy+in+pancreatic+cancer+cells+carrying+oncogenic+KRAS.">A novel E2F1-EP300-VMP1 pathway mediates gemcitabine-induced autophagy in pancreatic cancer cells carrying oncogenic KRAS.</a> Frontiers in Endocrinology. 11, 411 (2020).</li><li>Pardo, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gemcitabine+induces+the+VMP1-mediated+autophagy+pathway+to+promote+apoptotic+death+in+human+pancreatic+cancer+cells.">Gemcitabine induces the VMP1-mediated autophagy pathway to promote apoptotic death in human pancreatic cancer cells.</a> Pancreatology. 10 (1), 19-26 (2010).</li><li>Logsdon, C. D., Moessner, J., Williams, J. A., Goldfine, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoids+increase+amylase+mRNA+levels,+secretory+organelles,+and+secretion+in+pancreatic+acinar+AR42J+cells.">Glucocorticoids increase amylase mRNA levels, secretory organelles, and secretion in pancreatic acinar AR42J cells.</a> Journal of Cell Biology. 100 (4), 1200-1208 (1985).</li><li>Klionsky, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+autophagy+(4th+edition).">Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).</a> Autophagy. 17 (1), 1 (2021).</li><li>Segeritz, C. -. P., Vallier, L., Jalali, M., Saldanha, F. Y. L., Jalali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+9+-+Cell+culture:+Growing+cells+as+model+systems+in+vitro.">Chapter 9 - Cell culture: Growing cells as model systems in vitro.</a> Basic Science Methods for Clinical Researchers. , 151-172 (2017).</li><li>Elliott, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy:+Principles+and+modern+practices.">Confocal microscopy: Principles and modern practices.</a> Current Protocols in Cytometry. 92 (1), 68 (2020).</li><li>Grasso, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+novel+selective+autophagy+pathway+mediated+by+VMP1-USP9x-p62,+prevents+pancreatic+cell+death.">a novel selective autophagy pathway mediated by VMP1-USP9x-p62, prevents pancreatic cell death.</a> Journal of Biological Chemistry. 286 (10), 8308-8324 (2011).</li><li>Ropolo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pancreatitis-induced+vacuole+membrane+protein+1+triggers+autophagy+in+mammalian+cells.">The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells.</a> Journal of Biological Chemistry. 282 (51), 37124-37133 (2007).</li><li>Karanasios, E., Stapleton, E., Walker, S. A., Manifava, M., Ktistakis, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+of+early+autophagy+events:+Omegasomes+and+beyond.">Live cell imaging of early autophagy events: Omegasomes and beyond.</a> Journal of Visualized Experiments. (77), e50484 (2013).</li><li>Kuma, A., Matsui, M., Mizushima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC3,+an+autophagosome+marker,+can+be+incorporated+into+protein+aggregates+independent+of+autophagy:+caution+in+the+interpretation+of+LC3+localization.">LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization.</a> Autophagy. 3 (4), 323-328 (2007).</li><li>Zhang, Z., Singh, R., Aschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+detection+of+autophagy+in+mammalian+cells.">Methods for the detection of autophagy in mammalian cells.</a> Current Protocols in Toxicology. 69, 1-26 (2016).</li><li>Yoshii, S. R., Mizushima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+and+measuring+autophagy.">Monitoring and measuring autophagy.</a> International Journal of Molecular Sciences. 18 (9), 1865 (2017).</li><li>Betriu, N., Andreeva, A., Semino, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erlotinib+promotes+ligand-induced+EGFR+degradation+in+3D+but+not+2D+cultures+of+pancreatic+ductal+adenocarcinoma+cells.">Erlotinib promotes ligand-induced EGFR degradation in 3D but not 2D cultures of pancreatic ductal adenocarcinoma cells.</a> Cancers. 13 (18), 4504 (2021).</li><li>Wang, W., Dong, L., Zhao, B., Lu, J., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-cadherin+is+downregulated+by+microenvironmental+changes+in+pancreatic+cancer+and+induces+EMT.">E-cadherin is downregulated by microenvironmental changes in pancreatic cancer and induces EMT.</a> Oncology Reports. 40 (3), 1641-1649 (2018).</li><li>Kim, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+and+plasticity+of+cancer+cell+migration+in+a+pancreatic+tumor+three-dimensional+culture+model.">Phenotypic heterogeneity and plasticity of cancer cell migration in a pancreatic tumor three-dimensional culture model.</a> Cancers. 12 (5), 1305 (2020).</li><li>Meng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+EpCAM+over-expression+is+associated+with+favorable+clinical+outcomes+in+pancreatic+cancer+patients+with+Hepatitis+B+virus+negative+infection.">Cytoplasmic EpCAM over-expression is associated with favorable clinical outcomes in pancreatic cancer patients with Hepatitis B virus negative infection.</a> International Journal of Clinical and Experimental Medicine. 8 (12), 22204-22216 (2015).</li><li>Brock, R., Hamelers, I. H., Jovin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fixation+protocols+for+adherent+cultured+cells+applied+to+a+GFP+fusion+protein+of+the+epidermal+growth+factor+receptor.">Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor.</a> Cytometry. 35 (4), 353-362 (1999).</li></ol>

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, a...

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 c...

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 ...

The LC3 immunofluorescence enables the determination of autophagy levels in pancreatic cells. It permits the study of the potential effects of different agents on pancreatic cell autophagy. Immunofluorescence is a technique utilized to detect endogenous LC3 protein, which avoids problems caused by over-expressing LC3, such as forming protein aggregates unrelated to autophagy.Demonstrating the procedure will be Felipe Renna, a PhD graduate, and Malena Herrera Lopez, a PhD student from the laboratory of Maria Ines Vaccaro. To begin cell preparation, soak 12-millimeter round cover slips in absolute ethanol. After removing the cover, place the soaked cover slip vertically in the 24-well plate.Expose the multi-well plate to ultraviolet radiation for 15 minutes. Then position the cover slip horizontally and wash it with DMEM. Now see the low passage number of pancreatic cells, re-suspended in DMEM, containing 10%FBS, penicillin, and streptomycin, and incubate the cells for two days.Two days after cell seeding, prepare gemcitabine solution in DMEM at one microgram per microliter concentration. Then aspirate half the volume from each well in a separate tube, and add appropriate amount of gemcitabine solution. Return the half volume corresponding to each well back into the treated wells, and incubate the cells for 24 hours.For fixing and permeabilizing the cells, add cold methanol into a 24-well plate containing cells. Also prepare a six-well plate with cold PBS. Using a syringe needle and tweezers, take each cover slip and wash it twice in PBS.Then incubate in methanol for six minutes. After washing twice with PBS, incubate the cover slips in a blocking solution for one hour. Add an anti LC3 in the blocking solution at a ratio of one to 1000, and maintain on ice.Place a piece of laboratory sealing film over the multi-well lid, and add 25 microliters of anti-LC3 solution per cover slip over the sealing film. Using a syringe needle and tweezers, place each cover slip over the primary antibody drop, ensuring the cell side is in contact with the solution. Place the multi-well plate into the humidity chamber.Cover it with foil and incubate overnight in the fridge. The next day, remove the multi-well plate from the humidity chamber. Place the cover slips back on the multi-well plate and wash them with PBS three times.Dilute fluorescently-labeled anti-rabbit in blocking solution at a ratio of one to 800, and keep on ice in the dark. After sealing the multi-well lid, add 25 microliters of anti-rabbit solution per cover slip over the sealing film. And treat the cover slip with a secondary antibody as previously demonstrated.Then incubate the plate in the humidity chamber for two hours at room temperature, protected from light. At the end of incubation with antibody, wash the cover slips on the multi-well plate with PBS three times. Incubate each cover slip with the prepared DAPI solution for 10 minutes.Once washed with PBS, maintain the multi-well plate in the dark. For cell montage, prepare two beakers with water and a piece of paper. Add 10 microliters of PVA-DABCO solution per cover slip on a slide.Wash the cover slip in water, and then dry it on paper. Finally, place it over the PVA-DABCO drop. And dry overnight in the dark.The next day, using an inverted confocal microscope, visualize the labeled cells and capture the images. After capturing images, drag and drop each image file containing the captured channels in the Fiji screen. In the dialogue box, click OK to open the image.Then close the opened console window. From the Image tab, select Color, and then Split Channels. Close the image corresponding to the channels other than the LC3 image.Then in the Image tab, select Adjust, followed by Color Balance. Move the maximum slider to the left until the image saturates to visualize the cell contours. Use the freehand selection tool to draw the cell outline, and click reset to adjust the colors.To cut the selected item from the Edit tab, select Cut, and close the image without saving it. And from the Edit tab, select paste. In the Analyze menu, choose the tool 3D Objects Counter.Set the threshold at 2000, and the size filter at 50 to 500. Ensure the objects and summary boxes are marked, and click on OK.In the log window, the number of dots will be described as objects detected. The immunofluorescence indicated the cellular distribution of LC3 protein, whereas the DAPI showed nuclear localization in pancreatic cells.The LC3 dots significantly increased under gemcitabine treatment, indicating autophagic activity. Under an excessive confluence, the exocrine pancreas cells tend to pile up and grow on top of each other. The paraformaldehyde cell fixation method was ineffective for preserving LC3 proteins, as it did not reflect accurate distribution.When fixation or treatment was performed without waiting two days after seeding, the PANC1 cells had a round shape. This morphology decreased the relation between the cytoplasm and nucleus, making it difficult to understand the intracellular distribution of LC3. Incomplete methanol fixation could interfere with proper LC3 immuno labeling, leading to unclear images and suboptimal quantification.The most important thing to remember during this procedure is that the cells must be fixed in cold methanol for six minutes, instead of paraformaldehyde. Following this procedure, colocalization between LC3 and other proteins can be analyzed, allowing the study of different molecular pathways related to LC3 functions.

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Biology

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.

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

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 ...

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 ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view.
In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification.
In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.

Here, we present a protocol for optimized on-section correlative light-electron microscopy based on endogenous, fluorescent labeling as a tool to investigate the localization of rare proteins in relation to cellular ultrastructure. The power of this approach is demonstrated by ultrastructural localization of endogenous LC3 in starved cells without Bafilomycin treatment.

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal ...

Quantification of Mitochondrial Membrane Potential and Superoxide Levels

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

Author Spotlight: Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells  

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...