Name: assessing autophagic flux by measuring lc3, p62, and lamp1 co-localization using multispectral imaging flow cytometry
Uploaded: 2017-07-21T13:00:00
Duration: 11 min 39 s
Description: Watch this Scientific Journal Video about Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry at JoVE.com

I would like to thank my coworkers at MilliporeSigma, Philip J. Morrissey and Sherree L. Friend, for their support and guidance over the years. I would also like to thank Vidya Venkatachalam and Bryan R. Davidson for their help with the BDC3 feature in the IDEAS software, Ryan P. Jessup for help editing this manuscript, Raymond Kong for help on the day of shooting, and a special thank you to makeup artist Cynthia Xamonthiene.

<ol>
	<li>Levine, B., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+by+self-digestion:+molecular+mechanisms+and+biological+functions+of+autophagy.">Development by self-digestion: molecular mechanisms and biological functions of autophagy.</a> Developmental Cell. 6, 463-477 (2004).</li><li>Zhang, X. J., Chen, S., Huang, K. X., Le, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+should+autophagic+flux+be+assessed?.">Why should autophagic flux be assessed?.</a> Acta Pharmacologica Sinica. 34, 595-599 (2013).</li><li>Tanida, I., Takashi, U., Kominami, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC3+and+Autophagy.">LC3 and Autophagy.</a> Methods Mol Biol. 445, 77-88 (2008).</li><li>Larsen, K. B., Lamark, T., &#216;vervatn, A., Harneshaug, I., Johansen, T., Bj&#248;rk&#248;y, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reporter+cell+system+to+monitor+autophagy+based+on+p62/SQSTM1.">A reporter cell system to monitor autophagy based on p62/SQSTM1.</a> Autophagy. 6 (6), 784-793 (2010).</li><li>Demishtein, A., Porat, Z., Elazar, Z., Shvets, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+flow+cytometry+for+measurement+of+autophagy.">Applications of flow cytometry for measurement of autophagy.</a> Methods. 75, 87-95 (2015).</li><li>Mizushima, N., Yoshimori, T., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+mammalian+autophagy+research.">Methods in mammalian autophagy research.</a> Cell. 140 (3), 313-326 (2010).</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.">Guidelines for the use and interpretation of assays for monitoring autophagy.</a> Autophagy. 8 (4), 445-544 (2012).</li><li>Arsov, I., 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=BAC-mediated+transgenic+expression+of+fluorescent+autophagic+protein+Beclin+1+reveals+a+role+for+Beclin+1+in+lymphocyte+development.">BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development.</a> Cell Death Differ. 15 (9), 1385-1395 (2008).</li><li>Maloyan, A., Sayegh, J., Osinska, H., Chua, B. H., Robbins, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+death+pathways+in+desmin-related+cardiomyopathy.">Manipulation of death pathways in desmin-related cardiomyopathy.</a> Circ Res. 106 (9), 1524-1532 (2010).</li><li>Altman, B. 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=Autophagy+is+essential+to+suppress+cell+stress+and+to+allow+BCR-Abl-mediated+leukemogenesis.">Autophagy is essential to suppress cell stress and to allow BCR-Abl-mediated leukemogenesis.</a> Oncogene. 30 (16), 1855-1867 (2011).</li><li>Suarez, A. L., Kong, R., George, T., He, L., Yue, Z., van Dyk, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gammaherpesvirus+68+infection+of+endothelial+cells+requires+both+host+autophagy+genes+and+viral+oncogenes+for+optimal+survival+and+persistence.">Gammaherpesvirus 68 infection of endothelial cells requires both host autophagy genes and viral oncogenes for optimal survival and persistence.</a> J Virol. 85 (13), 6293-6308 (2011).</li><li>Tra, T., 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=Autophagy+in+human+embryonic+stem+cells.">Autophagy in human embryonic stem cells.</a> PLoS One. 6 (11), (2011).</li><li>Yuan, N., 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=Bafilomycin+A1+targets+both+autophagy+and+apoptosis+pathways+in+pediatric+B-cell+acute+lymphoblastic+leukemia.">Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia.</a> Haematologica. 100 (3), 345-356 (2015).</li><li>Leveque-El Mouttie, 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=Autophagy+is+required+for+stem+cell+mobilization+by+G-CSF.">Autophagy is required for stem cell mobilization by G-CSF.</a> Blood. 125 (19), 2933-2936 (2015).</li><li>Watson, A. S., 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=Autophagy+limits+proliferation+and+glycolytic+metabolism+in+acute+myeloid+leukemia.">Autophagy limits proliferation and glycolytic metabolism in acute myeloid leukemia.</a> Cell Death Discov. 1, 15008 (2015).</li><li>Stranks, A. 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=Autophagy+Controls+Acquisition+of+Aging+Features+in+Macrophages.">Autophagy Controls Acquisition of Aging Features in Macrophages.</a> J Innate Immun. 7 (4), 375-391 (2015).</li><li>Clarke, A. 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=Autophagy+is+activated+in+systemic+lupus+erythematosus+and+required+for+plasmablast+development.">Autophagy is activated in systemic lupus erythematosus and required for plasmablast development.</a> Ann Rheum Dis. 74 (5), 912-920 (2015).</li><li>Warnes, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+assays+for+the+study+of+autophagy.">Flow cytometric assays for the study of autophagy.</a> Methods. 82, 21-28 (2015).</li><li>Bj&#248;rk&#248;y, G., 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=p62/SQSTM1+forms+protein+aggregates+degraded+by+autophagy+and+has+a+protective+effect+on+huntingtin-induced+cell+death.">p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death.</a> J Cell Biol. 171 (4), 603-614 (2005).</li><li>Phadwal, 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=A+novel+method+for+autophagy+detection+in+primary+cells:+Impaired+levels+of+macroautophagy+in+immunosenescent+T+cells.">A novel method for autophagy detection in primary cells: Impaired levels of macroautophagy in immunosenescent T cells.</a> Autophagy. 8 (4), 677-689 (2012).</li><li>Rajan, R., Karbowniczek, M., Pugsley, H. R., Sabnani, M. K., Astrinidis, A., La-Beck, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+autophagosomes+and+autolysosomes+in+cells+using+imaging+flow+cytometry.">Quantifying autophagosomes and autolysosomes in cells using imaging flow cytometry.</a> Cytometry A. 87 (5), 451-458 (2015).</li><li>Kwok, A. S., 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=HspB8+mutation+causing+hereditary+distal+motor+neuropathy+impairs+lysosomal+delivery+of+autophagosomes.">HspB8 mutation causing hereditary distal motor neuropathy impairs lysosomal delivery of autophagosomes.</a> J Neurochem. 119 (6), 1155-1161 (2011).</li><li>Lopez-Herrera, G., 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=Deleterious+Mutations+in+LRBA+Are+Associated+with+a+Syndrome+of+Immune+Deficiency+and+Autoimmunity.">Deleterious Mutations in LRBA Are Associated with a Syndrome of Immune Deficiency and Autoimmunity.</a> Am J Hum Genet. 90 (6), 986-1001 (2012).</li><li>Pike, L. R., Phadwal, K., Simon, A. K., Harris, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATF4+orchestrates+a+program+of+BH3-only+protein+expression+in+severe+hypoxia.">ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia.</a> Mol Biol Rep. 39 (12), 10811-10822 (2012).</li><li>Pike, L. 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=Transcriptional+up-regulation+of+ULK1+by+ATF4+contributes+to+cancer+cell+survival.">Transcriptional up-regulation of ULK1 by ATF4 contributes to cancer cell survival.</a> Biochem J. 449 (2), 389-400 (2013).</li><li>Pugsley, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+autophagy:+Measuring+LC3+puncta+and+autolysosome+formation+in+cells+using+multispectral+imaging+flow+cytometry.">Quantifying autophagy: Measuring LC3 puncta and autolysosome formation in cells using multispectral imaging flow cytometry.</a> Methods. 112, 147-156 (2017).</li><li>IDEAS. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Image Data Exploration and Analysis Software User's Manual Version 6.2. , (2015).</li></ol>

I am employed by MilliporeSigma, the maker of the Amnis brand ImageStream, which was used in this study.

When performing this analysis, there are a few things to consider. It is important to have appropriate control samples. For this experiment, two different controls were used: Control and Control + Chloroquine. The Control sample was used to set the threshold for the regions. It represents the basal level of autophagy without stopping lysosomal degradation and is the control for the Starved sample. However, the Control sample is not an appropriate control to compare with the results from the Starved + Chloroquine sample because chloroquine itself influences the control sample. Therefore, it was necessary to have a Control + Chloroquine sample.
Prior to performing any analysis for autophagy, it is important to only analyze/gate on single cells that are in focus (i.e., gradient RMS greater than 60), as the results could be affected if there is more than one cell or the images are out of focus. It is crucial that the autophagosomes and lysosomes are in focus for proper spot counting and BDC3 analysis. Apoptotic cells must be removed prior to autophagy analysis, as they may skew results and should not be included. There should be appropriate staining for all three markers (i.e., LC3, p62, and LAMP1). For example, all three markers are intracellular and should have a punctate signal; if the signal is not punctate, or if it is on the surface of the cell, the staining would not be appropriate and the experiment/staining should be redone. In addition, for BDC3 to be accurate, it is essential to gate on cells that are bright for all three fluorescent markers of interest. Gating on positive events is needed to prevent the measurement of BDC3 on non-specific binding, imaging artifacts, and/or noise. This is crucial, as BDC3 can potentially amplify artifacts that are not true signals. Since BDC3 can only be performed on bright positive events, many cells may not be included in the final analysis; this is especially true for control cells that do not have any accumulation of LC3, p62, and/or LAMP1. Thus, a limitation of this assay is that BDC3 only examines cells that are positive for all three markers, which might not be appropriate for all autophagy experiments.
Like BDS, which has been previously reported15,16,17,20,21,22,23,24,25,26, BDC3 alone does not take into account the number of autophagy organelles that co-localize, resulting in a large degree of variability in the number of autophagosomes. Therefore, the bivariate approach presented by Rajan et al. was employed. Looking at the bivariate plots, one might ask if the cells must be positive for LC3, p62, and LAMP1; why are there so many cells that have 0 spots? This is because the LC3 signal might be bright enough for co-localization, but it might not meet the threshold set by the peak mask (Peak (M11, Ch11-LC3-AF647, Bright, 4)) necessary for it to be considered a spot.
The protocol presented here used MIFC to count LC3 puncta and the co-localization of three autophagy markers to measure autophagic flux. Under basal conditions (Control sample), the number of autophagosomes was low, and few cells were found with &#34;High Spots.&#34; With the addition of chloroquine, which inhibits autophagosome/lysosome fusion, there was an increase in the number of LC3 spots. Since the lysosome is unable to break down the autophagosome and the p62, which resides in the autophagosome, this leads to an increase in the co-localization of the LC3, p62, and LAMP1 autolysosome accumulation. This effect was amplified under nutrient starvation, which induces autophagy. However, without the addition of chloroquine, there was not a significant increase in the number of autophagosomes, most likely due to an increase in the rate of autophagic turnover. When cells were starved in the presence of chloroquine, there was an increase in the co-localization and number of autophagosomes, which supports the conclusion that starvation increases autophagic flux. EBSS is known to be a powerful inducer of autophagy. Therefore, it is expected that the increase would be large. If another method, such as drug induction, is used to induce autophagy, the difference between the control and treated samples may be subtler.
This particular protocol was designed to measure autophagy in human cell lines, but the assay could be adapted to other species by switching to antibodies for that particular species. In addition, the analysis method could be used for any intracellular application that requires the co-localization of three probes/markers.

Macroautophagy, hereafter referred to as autophagy, is a catabolic pathway in which damaged or dysfunctional components, long-lived proteins, and organelles are degraded via the lysosome and are recycled1. Autophagy is a dynamic, multistep process that involves the formation of an autophagosome; the fusion of the autophagosome with the lysosome, forming the autolysosome; and the degradation of the contents of the autolysosome2. The crucial biological marker used to identify autophagy in mammalian systems is the microtubule-associated protein 1A/1B-light chain 3 (LC3), which makes up the autophagosomal membrane. During autophagy, cytosolic LC3-1 is conjugated to phosphatidylethanolamine to form LC3-II; LC3-II is then incorporated into the autophagosomal membrane3. Another widely used marker for autophagic flux is the autophagy receptor sequestosome 1 (SQSTM1, p62) which physically links autophagic cargo to the autophagic membrane4,5.
Methods that have traditionally been used to measure autophagy are Western blots and fluorescence microscopy7. However, neither of these methods are considered to be the &#34;gold standard,&#34;2,6 as there are challenges associated with both of these techniques. Western blots only give an average because they use a homogenate sample, so observers cannot see what is happening in individual cells. On the other hand, fluorescence microscopy gives an observer information at the single-cell level, but it lacks throughput capabilities, making it difficult to obtain objective and statistically rigorous assessments. In recent years, the measurement of LC3 and p62 by multispectral imaging flow cytometry (MIFC, see the Table of Materials) has been gaining popularity due to its quantitative power, high throughput capabilities, multiplexing potential, and ability to image every cell. One of the most widely used methods to measure autophagy using MIFC, along with its companion analysis software (see the Table of Materials), is through the spot counting of LC3 puncta or autophagosomes8,9,10,11,12,13,14,15,16,17. However, an increase in autophagosomes does not necessarily mean that there is an increase in autophagy, as it could also represent a blockade in the process2. LC3-II turnover is a useful parameter to measure autophagic flux by analyzing cells in the presence and absence of a lysosomal degradation inhibitor, such as chloroquine. Chloroquine inhibits the fusion of the autophagosome to the lysosome, thereby permitting the quantitation of the autophagosome formation as a measure of the degree of autophagy by arresting autophagic flux before the lysosomal degradation can occur18. In addition, p62 is degraded primarily by autophagy, and if the lysosomal degradation of the autophagosome and its contents is blocked, an accumulation of p62 is expected17,19.
Autophagy is a multistep process, and the measurement of LC3 or p62 alone does not provide a complete picture of what is happening in the cells. Recent publications have emphasized the need to examine the concurrent formation of the autolysosome2,17,20,21. MIFC is uniquely able to measure the formation of the autolysosome by imaging the co-localization of the autophagosome to the lysosome15-17,20,21,22,23,24,25,26. In addition, the co-localization of p62 and LC3 using MIFC has also been explored to measure autophagic flux5. In the referenced analysis software, the &#34;feature&#34; that measures co-localization is called &#34;Bright Detail Similarity R3&#34; (BDS) and was specificallydesigned to compare the small, bright image details of two images. BDS is the log-transformed Pearson&#39;s correlation coefficient of the localized bright spots with a radius of 3 pixels or less; in other words, BDS calculates the degree of overlapping pixels from two different fluorescent channels. The bright spots are either correlated (i.e., same spatial location) or uncorrelated (i.e., different spatial locations). Therefore, the correlation coefficient varies between 0 (uncorrelated) and 1 (perfect correlation). The coefficient is log-transformed to increase the range to between zero and infinity26,27. BDS alone may not be sufficient; Rajan et al. found that using only BDS could lead to false-positive or false-negative results21. BDS evaluates the co-localization of two markers of autophagy but does not consider the number of autophagosomes. To account for the number of autophagosomes, Rajan et al. included spot-counting of the LC3 puncta21. Rajan et al. proposed using a bivariate scatter plot of LC3 spot count versus BDS. Using this bivariate plot, two populations were first identified: one with cells that have a high level of LC3 spots and one with cells that have a low level of LC3 spots. The high-LC3 spot population was further classified into two populations: cells with low co-localization (i.e., accumulation of autophagosomes) and cells with high co-localization (i.e., accumulation of autolysosomes). This bivariate plot allows one to distinguish between cells with very few autophagosomes and cells with an accumulation of autophagosomes and/or autolysosomes21.
Until now, the simultaneous co-localization of LC3, p62, and LAMP1 (lysosomal marker) was not possible using a single &#34;Feature Type&#34; in the MIFC companion analysis software (see the Table of Materials). However, a new feature, recently introduced in version 6.1, is called Bright Detail Colocalization 3 (BDC3). BDC3 compares the bright detail images from each of the three images (in this case, LC3, p62, and LAMP1) and quantifies the co-localization of the three probes (i.e., LC3, p62, and LAMP1). The BDC3 feature computes the Pearson&#39;s correlation coefficient modified to extend to three images. Since the bright spots in the three images are either correlated or uncorrelated, the correlation coefficient varies between 0 (uncorrelated) and 1 (perfect correlation). The coefficient is log-transformed to increase the dynamic range between 0 and infinity. By switching out the BDS feature with the BDC3 feature, the analysis method presented by Rajan et al. can now incorporate the three most-used markers to measure autophagy in one system at the same time. The ability to co-localize these three markers of autophagy in a single assay could lead to novel insights into the induction and regulation of autophagy. The following protocol outlines the steps to induce autophagy in Jurkat cells; label the cells with LC3, p62, and LAMP1 antibodies; acquire data on a multispectral imaging flow cytometer; and analyze the data to assess autophagic flux.

<table border="0" cellpadding="0" cellspacing="0" width="1012">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">15 mL centrifuge tube</td>
			<td style="width:244px;">Falcon</td>
			<td style="width:161px;">352096</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">50 mL centrifuge tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;">AF647 labeled Donkey anti-Mouse - IgG&#160;</td>
			<td>Invitrogen</td>
			<td>A-31571</td>
			<td style="width:299px;">Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647. Concntration 2mg/mL. https://www.thermofisher.com/antibody/product/Donkey-anti-Mouse-IgG-H-L-Secondary-Antibody-Polyclonal/A-31571</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:308px;">anti-human CD107a-PE (LAMP1)</td>
			<td>BioLegend</td>
			<td>328607</td>
			<td style="width:299px;">Clone H4A3. Concentration 400 &#181;g/mL. http://www.biolegend.com/pe-anti-human-cd107a-lamp-1-antibody-4967.html</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;">anti-human CD45- AF488</td>
			<td>BioLegend</td>
			<td>304017</td>
			<td style="width:299px;">Clone HI30. http://www.biolegend.com/alexa-fluor-488-anti-human-cd45-antibody-2738.html</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">anti-human CD45- PE&#160;</td>
			<td>BioLegend</td>
			<td>304008</td>
			<td style="width:299px;">Clone HI30.&#160; http://www.biolegend.com/pe-anti-human-cd45-antibody-708.html</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;">anti-human CD45-AF647&#160;</td>
			<td>BioLegend</td>
			<td>304018</td>
			<td style="width:299px;">Clone HI30.&#160; http://www.biolegend.com/alexa-fluor-647-anti-human-cd45-antibody-2739.html</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Anti-LC3 (Human) mAb clone 4E12</td>
			<td>MBL International Corporation</td>
			<td>M152-3</td>
			<td style="width:299px;">Clone 4E+12.&#160; Concentration 2 mg/mL.&#160; https://www.mblintl.com/products/m152-3</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:308px;">Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (Alexa Fluor 488)</td>
			<td>abcam</td>
			<td>ab185015</td>
			<td style="width:299px;">Clone EPR4844.&#160; Concentration 0.5 mg/mL.&#160; http://www.abcam.com/sqstm1-p62-antibody-epr4844-autophagosome-marker-alexa-fluor-488-ab185015.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Chloroquine diphosphate Salt</td>
			<td>Sigma-Aldrich</td>
			<td>C6628-25G</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:308px;">Cleanser - Coulter Clenz&#160;</td>
			<td>Beckman Coulter</td>
			<td>8546931</td>
			<td style="width:299px;">Fill container with 200 mL of Cleanser.&#160; https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=8546931#2/10//0/25/1/0/asc/2/8546931///0/1//0/</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:308px;">DAPI Stain 5mg/mL</td>
			<td>MilliporeSigma</td>
			<td>508741</td>
			<td style="width:299px;">http://www.emdmillipore.com/US/en/product/DAPI-Stain---CAS-28718-90-3---Calbiochem,EMD_BIO-508741</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:308px;">Debubbler - 70% Isopropanol</td>
			<td>MilliporeSigma</td>
			<td>1.3704</td>
			<td style="width:299px;">Fill container with 200 mL of Debubbler.&#160; http://www.emdmillipore.com/US/en/product/2-Propanol-70%25-%28V%2FV%29-0.1-%C2%B5m-filtred,MDA_CHEM-137040?ReferrerURL=https%3A%2F%2Fwww.google.com%2F</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Dulbecco&#39;s Phosphate Buffered Saline 10X</td>
			<td>MilliporeSigma</td>
			<td>BSS-2010-B</td>
			<td style="width:299px;">&#160;Ca++Mg++ Free&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Dulbecco&#39;s Phosphate Buffered Saline 1X</td>
			<td>MilliporeSigma</td>
			<td>BSS-1006-B</td>
			<td style="width:299px;">PBS Ca++Mg++ Free&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Earle&#39;s Balanced Salt Solution 1X</td>
			<td>Gibco</td>
			<td>14155</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Fetal Bovine Serum</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="241">
			<td height="241" style="height:241px;width:308px;">Formaldehyde, 10%, methanol free, Ultra Pure</td>
			<td>Polysciences, Inc.</td>
			<td>04018</td>
			<td style="width:299px;">This is what is used for the 4% and 1% Formalin. CAUTION: Formalin/Formaldehyde toxic by inhalation and if swallowed.&#160; Irritating to the eyes, respiratory systems and skin.&#160; May cause sensitization by inhalation or skin contact. Risk of serious damage to eyes.&#160; Potential cancer hazard.&#160; http://www.polysciences.com/default/catalog-products/life-sciences/histology-microscopy/fixatives/formaldehydes/formaldehyde-10-methanol-free-pure/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Hanks&#39; Balanced Salt Solution 1X</td>
			<td>Gibco</td>
			<td>14175</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Jurkat, Clone E6-1 (ATCC TIB-152)</td>
			<td>ATCC</td>
			<td>TIB-152</td>
			<td style="width:299px;">https://www.atcc.org/products/all/TIB-152.aspx</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">MEM Non-Essential Amino Acids 100X</td>
			<td>HyClone</td>
			<td>SH30238.01</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="201">
			<td height="201" style="height:201px;width:308px;">MIFC - ImageStreamX Mark II</td>
			<td>MilliporeSigma</td>
			<td>100220</td>
			<td style="width:299px;">A 2 camera ImageStreamX Mark II eqiped with the 405nm, 488nm, and 642nm lasers was used. http://www.emdmillipore.com/US/en/life-science-research/cell-analysis/amnis-imaging-flow-cytometers/imagestreamx-Mark-ii-imaging-flow-cytometer/VaSb.qB.QokAAAFLzRop.zHe,nav?cid=BI-XX-BDS-P-GOOG-FLOW-B325-0006</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:308px;">MIFC analysis software - IDEAS 6.2</td>
			<td>MilliporeSigma</td>
			<td>100220</td>
			<td style="width:299px;">The companion software to the MIFC (ImageStreamX MKII).&#160; IDEAS version 6.2</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:308px;">MIFC software - INSPIRE</td>
			<td>MilliporeSigma</td>
			<td>100220</td>
			<td style="width:299px;">This is the software that runs the MIFC (ImageStreamX MKII). INSPIRE version 200.1.388.0</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">PE anti-human CD107a (LAMP-1) Antibody clone H4A3</td>
			<td>BioLegend</td>
			<td>328608</td>
			<td style="width:299px;">http://www.biolegend.com/pe-anti-human-cd107a-lamp-1-antibody-4967.html</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Penicllin/Streptomycin/Glutamine solution 100X</td>
			<td>HyClone</td>
			<td>SV30082.1</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:308px;">Rinse - Ultrapure water or deionized water</td>
			<td>NA</td>
			<td>NA</td>
			<td style="width:299px;">You can use any ultrapure water or deionized water.&#160; Fill container with 900 mL of Rinse.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">RPMI-1640 Medium 1X</td>
			<td>HyClone</td>
			<td>SH30027.01</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:308px;">Sheath - PBS</td>
			<td>MilliporeSigma</td>
			<td>BSS-1006-B</td>
			<td style="width:299px;">This is the same as Dulbecco&#39;s Phosphate Buffered Saline 1X&#160; Ca++MG++ free.&#160; Fill container with 900mL of Sheath.</td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:308px;">Siliconized polypropylene microcentrifuge tubes</td>
			<td>Fisherbrand</td>
			<td>02-681-320</td>
			<td style="width:299px;">Fisherbran Siliconized Low-Retention Microcentrifuge Tubes 1.5 mL. https://www.fishersci.com/shop/products/fisherbrand-siliconized-low-retention-microcentrifuge-tubes-8/p-193936</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Sodium Pyruvate solution (100mM)</td>
			<td>HyClone</td>
			<td>SH30239.01</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:308px;">Sterilizer - 0.4-0.7% Hypochlorite</td>
			<td>VWR</td>
			<td>JT9416-1</td>
			<td style="width:299px;">This is assentually 10% Clorox bleach that can be made by deluting Clorox bleach with water.&#160; Fill container with 200 mL of Sterilzer.</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:308px;">System Calibration Reagent - SpeedBead</td>
			<td>MilliporeSigma</td>
			<td>400041</td>
			<td style="width:299px;">Each tube holds ~10 mL.&#160; https://www.emdmillipore.com/US/en/life-science-research/cell-analysis/amnis-imaging-flow-cytometers/support-training/XDqb.qB.wQMAAAFLBDUp.zHu,nav&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">T75 flask</td>
			<td>Falcon</td>
			<td>353136</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:308px;">TRITON X-100, PROTEIN GRADE Detergent, 10% Solution, Sterile-Filtered</td>
			<td>MilliporeSigma</td>
			<td>648463-50ML</td>
			<td style="width:299px;">http://www.emdmillipore.com/US/en/product/TRITON-X-100,-PROTEIN-GRADE-Detergent,-10%25-Solution,-Sterile-Filtered---CAS-9002-93-1---Calbiochem,EMD_BIO-648463</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Water, Cell Culture Grade&#160;</td>
			<td>HyClone</td>
			<td>SH30529.03</td>
			<td style="width:299px;"></td>
		</tr>
	</tbody>
</table>

assessing autophagic flux by measuring lc3, p62, and lamp1 co-localization using multispectral imaging flow cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

This analysis method uses multiple features to asses autophagic flux. In order to fully understand the final bivariate plot, the individual analysis features must first be investigated. The counting of autophagosomes is a logical way to measure autophagy; however, the size/shape/brightness of LC3 puncta can vary drastically between cells. Variation can make it difficult to count autophagosomes manually or using a spot-count feature in the analysis software. Therefore, no spot-count feature will be perfect due to this large variability in autophagosomes. However, a good spot-count feature will work on most cells26. Figure 3 shows examples of spot masking of LC3 puncta in Jurkat cells using different spot masks. The spot-count feature selected for this data set is Spot Count_Peak(M11, Ch11-LC3-AF647, Bright, 4)_4, meaning that the spot-count feature counted the spots that the peak mask identified within the default channel 11 mask (M11) on channel 11 (Ch11-LC3-AF647) with a spot-to-cell background ratio of 4 (Bright, 4). Figure 4 shows spot-count histograms and representative images for the mean spot count for the Control, Control + Chloroquine, Starved, and Starved + Chloroquine Jurkat cells labeled with anti-LC3-AF647. The Control and Starved mean spot counts are not significantly different; with the addition of chloroquine, there is a large difference in the mean of the Control + Chloroquine compared to the Starved + Chloroquine.
The next feature to investigate is BDC3. BDC3 is a measurement of the co-localization of three markers/probes, in this case, LC3, p62, and LAMP1. Figure 5A-5D shows BDC3 histograms for the Control, Control + Chloroquine, Starved, and Starved + Chloroquine Jurkat cells labeled with anti-p62-AF488, anti-LAMP1-PE, and anti-LC3-AF647. There is a shift between the Control mean to the Starved mean, as well as the Control + Chloroquine mean to the Starved + Chloroquine mean. However, looking at the images of cells from the mean BDC3 scores in Figure 5E-5H, there is a greater difference between the samples than the histograms may lead one to believe. This is because BDC3 does not consider the number of autophagy organelles that co-localize, resulting in a large degree of variability in the number of autophagosomes for the same BDC3 score. In most cases, there is overlap between all three probes because, even at basal levels, p62, LAMP1, and LC3 should, to a certain extent, co-localize or reside in similar regions in the cells. As a contrast, an example of three probes that should not co-localize are anti-p62-AF488, anti-LC3-AF647, and DAPI nuclear dye for the Starved + Chloroquine sample, shown in Figure 6.
When the spot-count feature and the BDC3 feature are combined, the presence of different subpopulations that improve the ability to distinguish between the various samples/conditions are evident. Figure 7 shows the bivariate plot of spot count of LC3 versus BDC3 p62/LAMP1/LC3 for the four samples: Control, Control + Chloroquine, Starved, and Starved + Chloroquine. The Control sample was used to set the gating strategy for three populations: Low Spots, High Spots/Low BDC3, and High Spots/High BDC3. The Control samples demonstrated that greater than 98% of the cells had 1 or fewer spots. The boundary between the High Spots/Low BDC3 and High Spots/High BDC3 was set to a BDC3 score of 1 because more than 91% of the Control sample had a BDC3 score of less than 1. A summary of the results for the bivariate plots is shown in Table 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55637/55637fig1.jpg" /> 
Figure 1: MIFC Instrument Setting. A screenshot of the MIFC instrument settings used for this experiment, outlined in step 8 of the protocol. <a href="http://cloudflare.jove.com/files/ftp_upload/55637/55637fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55637/55637fig2.jpg" /> 
Figure 2: Analysis Software Gating Strategy. A screenshot of the analysis software gating scheme, outlined in step 9 of the protocol. <a href="http://cloudflare.jove.com/files/ftp_upload/55637/55637fig2large.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/55637/55637fig3.jpg" /> 
Figure 3: LC3 Spot Masking. Jurkat cell images and masks used to create the spot-count feature. Shown are BrightField (BF), LC3-AF647, Peak(M11, Ch11-LC3-AF647, Bright,2), Peak(M11, Ch11-LC3-AF647, Bright,4), Peak(M11, Ch11-LC3-AF647, Bright,5), Spot(M11, Ch11-LC3-AF647, Bright,5,3,1), and Spot(M11, Ch11-LC3-AF647, Bright,6,2,1). The mask that worked best for all cells shown was Peak(M11, Ch11-LC3-AF647, Bright,4). <a href="http://cloudflare.jove.com/files/ftp_upload/55637/55637fig3large.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/55637/55637fig4.jpg" /> 
Figure 4: LC3 Spot-count Histograms. Using the spot-count feature Spot Count_Peak(M11, Ch11-LC3-AF647, Bright, 4)_4 and the LC3-AF647 spot-count histograms for Control (A, light blue), Control + Chloroquine (B, blue), Starved (C, pink), and Starved + Chloroquine (D, red). The mean spot counts for Control, Control + Chloroquine, Starved, and Starved + Chloroquine are 0.07, 1.13, 0.10, and 2.77, respectively. BF, LC3-AF647 (red), DAPI nuclear dye (blue), and a composite of the LC3-AF647 and DAPI images of representative cells for the mean spot count are shown for Control (E, 0 spots), Control + Chloroquine (F, 1 spot), Starved (G, 0 spots), and Starved + Chloroquine (H, 3 spots). <a href="http://cloudflare.jove.com/files/ftp_upload/55637/55637fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55637/55637fig5.jpg" /> 
Figure 5: BDC3 Histograms of p62/LAMP1/LC3. BDC3 p62/LAMP1/LC3 histograms for Control (A, light blue), Control + Chloroquine (B, blue), Starved (C, pink), and Starved + Chloroquine (D, red). The mean BDC3 score for Control, Control + Chloroquine, Starved, and Starved + Chloroquine are 0.57, 0.82, 0.74, and 0.98, respectively. BF; p62-AF488 (green); LAMP1-PE (yellow); LC3-AF647 (red); and a composite of the p62-AF488, LAMP1-PE, and LC3-AF647 images of representative cells for the mean BDC3 are shown for Control (E), Control + Chloroquine (F), Starved (G), and Starved + Chloroquine (H). <a href="http://cloudflare.jove.com/files/ftp_upload/55637/55637fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55637/55637fig6.jpg" /> 
Figure 6: BDC3 Histograms of p62/LC3/DAPI for the Starved + Chloroquine Sample. (A) BDC3 p62/LC3/DAPI histogram for the Starved + Chloroquine sample is shown. The mean BDC3 score is 0.07. (B) BF; p62-AF488 (green); LC3-AF647 (red); DAPI (blue); and a composite of the p62-AF488, LC3-AF647, and DAPI images of representative cells for the mean BDC3 is shown for the Starved + Chloroquine sample. <a href="http://cloudflare.jove.com/files/ftp_upload/55637/55637fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55637/55637fig7.jpg" /> 
Figure 7: Bivariate Plots of LC3 Spot Count versus BDC3 p62/LAMP1/LC3. (A) Bivariate plots of LC3 spot count versus BDC3 p62/LAMP1/LC3 for Control, Control + Chloroquine, Starved, and Starved + Chloroquine Jurkat cells. (B) BF; p62-AF488 (green); LAMP1-PE (yellow); LC3-AF647 (red); and a composite of the p62-AF488, LAMP1-PE, and LC3-AF647 images from three regions (i.e., Low Spots, High Spots/Low BDC3, and High Spots/High BDC3) are shown for the Starved + Chloroquine sample. <a href="http://cloudflare.jove.com/files/ftp_upload/55637/55637fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Cell Count</td>
			<td>Bright Detail Colocalization 3 Mean&#160;</td>
			<td>Spot Count LC3 Mean</td>
			<td>% Low Spot&#160;&#160;</td>
			<td>% High Spot/Low BDC3&#160;</td>
			<td>% High Spot/High BDC3&#160;</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>439</td>
			<td>0.57</td>
			<td>0.07</td>
			<td>98.2</td>
			<td>1.8</td>
			<td>0.0</td>
		</tr>
		<tr>
			<td>Control + Chloroquine</td>
			<td>1432</td>
			<td>0.82</td>
			<td>1.13</td>
			<td>68.9</td>
			<td>19.5</td>
			<td>11.7</td>
		</tr>
		<tr>
			<td>Starved&#160;</td>
			<td>1204</td>
			<td>0.74</td>
			<td>0.10</td>
			<td>98.4</td>
			<td>0.6</td>
			<td>1.0</td>
		</tr>
		<tr>
			<td>Starved + Chloroquine</td>
			<td>1811</td>
			<td>0.98</td>
			<td>2.77</td>
			<td>32.1</td>
			<td>36.9</td>
			<td>31.0</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of Jurkat LC3 Spot Count versus BDC3 p62/LAMP1/LC3 Bivariate Plot

Watch this Scientific Journal Video about Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry at JoVE.com

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Research

JoVE Journal

Biology

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...