Name: the lactate dehydrogenase sequestration assay &#8212; a simple and reliable method to determine bulk autophagic sequestration activity in mammalian cells
Uploaded: 2018-07-27T14:30:00
Description: Watch this Scientific Journal Video about The Lactate Dehydrogenase Sequestration Assay â€” A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells at

This work was financially supported by the Research Council of Norway, the University of Oslo, the Anders Jahre Foundation, the Nansen Foundation, and the Legacy in the memory of Henrik Homan. We thank Dr. Noboru Mizushima for the ATG5+/+ MEFs and ATG5-/- MEFs, Dr. Masaaki Komatsu for the ATG7+/+ MEFs and ATG7-/- MEFs, and Dr. Shizuo Akira for the ATG9A+/+ MEFs and ATG9A-/- MEFs. We thank Frank S&#230;tre for technical assistance, and Dr. Per O. Seglen for constructive methodological discussions.

1. Cell Seeding and Treatment
<ol>
	<li>Culture adherent cells in 75 cm2 tissue culture flasks in a humidified incubator with 5% CO2 at 37 &#176;C, using the preferred culture medium for the cell type in question. Allow the cells to grow until they reach a near-confluent cell layer. 
	NOTE: Use RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) for LNCaP, HEK293, mouse embryonic fibroblasts (MEFs), BJ, MCF-7, and RPE-1 cells.
	<ol>
		<li>Wash the cells with 3 mL&...

In summary, the protocol described here represents a reliable and widely applicable method to monitor bulk autophagic sequestration activity in mammalian cells. Compared to the original method12,16, we have removed a number of unnecessary steps, simplified several of the remaining steps, and introduced a substantial downscaling. As a result, the protocol is greatly improved in relation to cost- and time-efficiency, and the same amount of samples can now be handle...

Autophagy (Greek for &#34;self-eating&#34;) is an evolutionary conserved process for vacuolar/lysosomal degradation of intracellular material. Upon discovery of the autophagy-related (&#34;ATG&#34;) genes, which are important for autophagy in yeast and humans, and the realization that autophagy plays a significant role in human health and disease (acknowledged by the 2016 Nobel Prize in Medicine or Physiology to Yoshinori Ohsumi), autophagy has quickly become one of the most intensely studied processes in cell biology1,2.
Macroautophagy (hereafter referred to as &#34;autophagy&#34;) is characterized by the expansion and folding of intracellular membrane cisternae (&#34;phagophores&#34;) into sealed, double- or multi-membrane structures (&#34;autophagosomes&#34;) that effectively sequester the enwrapped material from the rest of the cytoplasm. Upon fusion of autophagosomes with lysosomes, the inner autophagosomal membrane and the sequestered cargo is degraded and recycled. Autophagosomes can sequester cytoplasmic material in both random (non-selective autophagy) and selective (selective autophagy) manners. Bulk autophagy most likely represents a mix of non-selective and selective autophagy.
In the 1960&#39;s and 70&#39;s (&#34;the morphological era&#34; of autophagy research), autophagic sequestration was mainly assessed through ultrastructural analyses. In the 1980&#39;s and beginning of the 1990&#39;s (&#34;the biochemical era&#34;) Per Seglen and co-workers &#8212; who studied autophagy in primary rat hepatocytes &#8212; developed the first methods to quantitatively measure autophagic sequestration activity3. Using these assays, Seglen defined and characterized different steps of the autophagic-lysosomal pathway4,5, discovered and coined the amphisome6 (the product of endosome-autophagosome fusion), and was the first to describe the role of protein phosphorylation in autophagy regulation7. However, after the discovery of the ATGs in the 1990&#39;s (&#34;the molecular&#160;era&#34;) and the first characterization of a mammalian ATG8 protein, microtubule-associated protein 1A/1B-light chain 3 (LC3) in 20008, the use of ATG proteins as markers for the autophagic process quickly gained popularity, and the older and more laborious biochemical methods were left behind. In fact, over the last 18 years, western blot and fluorescence microscopy analyses of LC3 have become the by far most popular (and in many cases the only) means of studying autophagy in mammalian cells. The advantage is the relative ease by which these methods can be carried out. The disadvantage is that one is studying a cart component (LC3) rather than actual autophagic cargo. This is a rather serious disadvantage, because the relationship between the states and/or flux of LC3 through the pathway versus the sequestration and flux of cargo is highly unclear. In fact, we have shown that bulk cargo flux can be maintained at high levels under conditions where there is no LC3 flux, despite the presence of conjugated LC3 in the cells9. Moreover, we demonstrated that bulk autophagy is unaffected by efficient LC3 depletion, and thus likely is LC3-independent9. This finding has later been confirmed by LC3 knock-out studies10,11, which also indicate that Parkin-dependent mitophagy (the selective autophagy of mitochondria) is independent of LC310,11.
In summary, there is a clear need for cargo-based assays to monitor autophagic activity. Optimally such assays should be broadly applicable, well-defined, and easy to perform. Over the last few years we have taken a particular interest in the LDH sequestration assay, which was developed by Per Seglen in the 1980&#39;s12, and is based on measuring the transfer of cytosolic LDH to sedimentable, autophagic vacuole-containing cell fractions. LDH is a stable, soluble cytosolic protein that is readily co-sequestered when phagophores enwrap cytoplasmic cargo. Sequestration of LDH is therefore a general measure of autophagic sequestration. LDH is exclusively degraded by the autophagic-lysosomal pathway12. Thus, in the presence of lysosomal degradation inhibitors, e.g., bafilomycin A1 (Baf)13, experimental treatment effects directly reflect alterations in autophagic sequestration activity. In the absence of degradation inhibitors, the net effect of alterations in LDH sequestration and degradation can be measured.
The LDH sequestration assay is broadly applicable, since LDH is highly and ubiquitously expressed in all cell types, and LDH levels can be accurately quantified by an enzymatic assay14,15. However, the original protocol12 &#8212; established in primary rat hepatocytes &#8212; was rather time-consuming and required a high amount of starting material as well as a custom-made electric discharge capacitor. In a step-wise manner, we have gradually transformed the assay into an easy and versatile method. First, the original protocol was adapted for use in mammalian cell lines16. Second, the method was substantially downscaled3,9. Third, several steps in the protocol were eliminated, including a laborious density cushion step17. This simultaneously enabled an even further downscaling of the method, from the original starting point of using a 10 cm plate per sample16 to using a single well from a 12-well plate per sample (i.e., approximately 15-fold less starting material)17. Fourth, we identified a commercial electroporation apparatus that could replace the custom-made electric discharge capacitor17.
Here our most up-to-date protocol of the LDH sequestration assay, which includes some further simplifications of the method as compared to the previously published17 is presented. Furthermore, a set of typical results obtained in a number of different cell types is shown, and importantly, multiple lines of experimental validations of the method using pharmacological as well as genetic knockdown and knockout approaches are provided. For an overall flow scheme of the whole protocol, see Figure 1.

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		<tr height="40">
			<td height="40" style="height:40px;width:487px;">1.5 ml and 2 ml microcentrifuge tubes&#160;</td>
			<td style="width:221px;">Eppendorf</td>
			<td style="width:159px;">211-2130 and 211-2120</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">12-well plates&#160;</td>
			<td style="width:221px;">Falcon</td>
			<td style="width:159px;">353043</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Accumax&#160; cell detachment solution</td>
			<td style="width:221px;">Innovative Cell Technologies</td>
			<td style="width:159px;">A7089</td>
			<td>Keep aliquots at -20 &#176;C for years, and in fridge for a few months</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Bafilomycin A1</td>
			<td style="width:221px;">Enzo</td>
			<td style="width:159px;">BML-CM110-0100</td>
			<td>Dissolve in DMSO</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">BJ cells</td>
			<td style="width:221px;">ATCC</td>
			<td style="width:159px;">CRL-2522</td>
			<td>use at passage &#60;30</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Bovine serum albumin (BSA)</td>
			<td style="width:221px;">VWR</td>
			<td style="width:159px;">422361V</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Burker counting chamber</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:159px;">139-658585</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Countess Cell Counting Chamber Slides</td>
			<td style="width:221px;">ThermoFisher Scientfic</td>
			<td style="width:159px;">C10228</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Countess II Automated Cell Counter</td>
			<td style="width:221px;">ThermoFisher Scientfic</td>
			<td style="width:159px;">AMQAX1000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Cover glass for the Burker counting chamber</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:159px;">139-658586</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Criterion Tris-HCl Gel, 4&#8211;20%, 26-well, 15 &#181;l, 13.3 x 8.7 cm (W x L)&#160;</td>
			<td style="width:221px;">Bio-Rad</td>
			<td style="width:159px;">3450034</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">DTT</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:159px;">D0632</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Earle&#39;s balanced salt solution (EBSS)</td>
			<td style="width:221px;">Gibco</td>
			<td style="width:159px;">24010-043</td>
			<td>conatains 0.1% glucose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">EDTA</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:159px;">E7889</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Electroporation cuvette (4 mm)</td>
			<td style="width:221px;">Bio-Rad</td>
			<td style="width:159px;">1652088</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Exponential decay wave electroporator</td>
			<td style="width:221px;">BTX Harvard Apparatus</td>
			<td style="width:159px;">&#160;EMC 630</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Fetal bovine serum (FBS)</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">F7524</td>
			<td style="width:703px;">10% final concentration in RPMI 1640 medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">HEK293 cells</td>
			<td style="width:221px;">ATCC</td>
			<td style="width:159px;">CRL-1573</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Imidazole</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:159px;">56750</td>
			<td>Autoclave a 65 mM solution and keep in fridge for months</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Incubator; Autoflow IR Direct Heat CO2 incubator</td>
			<td style="width:221px;">NuAire</td>
			<td style="width:159px;">NU-5510E</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Lipofectamine RNAiMAX Transfection Reagent</td>
			<td style="width:221px;">ThermoFisher</td>
			<td style="width:159px;">13778150</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">LNCaP cells</td>
			<td style="width:221px;">ATCC</td>
			<td style="width:159px;">CRL-1740</td>
			<td>use at passage &#60;30</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">3-Methyl Adenine (3MA)</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:159px;">M9281</td>
			<td>Stock 100 mM in RPMI in -20 &#176;C.&#160; Heat stock to 65 &#176;C for 10 minutes, and use at 10 mM final concentration</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Refridgerated Microcentrifuge</td>
			<td style="width:221px;">Beckman Coulter Life Sciences</td>
			<td style="width:159px;">368831</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Refridgerated Microcentrifuge with soft-mode function</td>
			<td style="width:221px;">Eppendorf&#160;</td>
			<td>Eppendorf 5417R&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">MRT67307 hydrochloride (ULKi)</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:159px;">SML0702</td>
			<td>Inhibits ULK kinase activity. Dissolve in DMSO.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">MaxMat Multianalyzer instrument</td>
			<td style="width:221px;">Erba Diagnostics</td>
			<td style="width:159px;">PL-II</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">MCF7 cells</td>
			<td style="width:221px;">ATCC</td>
			<td style="width:159px;">HTB-22</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">NADH</td>
			<td style="width:221px;">Merck-Millipore</td>
			<td style="width:159px;">1.24644.001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Nycodenz</td>
			<td style="width:221px;">Axis-Shield</td>
			<td style="width:159px;">1002424</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Opti-MEM Reduced Serum Medium</td>
			<td style="width:221px;">ThermoFisher</td>
			<td style="width:159px;">31985062</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Phosphate-buffered saline (PBS)</td>
			<td style="width:221px;">Gibco</td>
			<td style="width:159px;">20012-019</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Pipette tips 3 (0.5-20 &#181;l)</td>
			<td style="width:221px;">VWR</td>
			<td style="width:159px;">732-2223</td>
			<td>Thermo Fischer ART Barrier tips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Pipette tips (1-200 &#181;l)</td>
			<td style="width:221px;">VWR</td>
			<td style="width:159px;">732-2207&#160;</td>
			<td>Thermo Fischer ART Barrier tips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Pipette tips (100-1000&#181;l)</td>
			<td style="width:221px;">VWR</td>
			<td style="width:159px;">732-2355&#160;</td>
			<td>Thermo Fischer ART Barrier tips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Pipettes</td>
			<td style="width:221px;">ThermoFisher</td>
			<td style="width:159px;">4701070</td>
			<td>Finnpipette F2 GLP Kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Poly-D-lysine</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:159px;">P6407-10X5MG</td>
			<td>Make a 1 mg/ml stock solution in sterile H2O. This solution is stable at -20 &#176;C for at least 1 year.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Pyruvate</td>
			<td>Merck-Millipore</td>
			<td>1066190050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">RPE-1 cells (hTERT RPE-1)</td>
			<td style="width:221px;">ATCC</td>
			<td style="width:159px;">CRL-4000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">RPMI 1640</td>
			<td style="width:221px;">Gibco</td>
			<td style="width:159px;">21875-037</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">SAR-405</td>
			<td style="width:221px;">ApexBio&#160;</td>
			<td style="width:159px;">A8883</td>
			<td style="width:703px;">Inhibits phosphoinositide 3-kinase class III (PIK3C3). Dissolve in DMSO.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Silencer Select Negative Control #1 (siCtrl)</td>
			<td style="width:221px;">ThermoFisher/Ambion</td>
			<td style="width:159px;">4390843</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Silencer Select ATG9-targeting siRNA (siATG9A)</td>
			<td style="width:221px;">ThermoFisher/Ambion</td>
			<td style="width:159px;">s35504</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Silencer Select FIP200-targeting siRNA (siFIP200)</td>
			<td style="width:221px;">ThermoFisher/Ambion</td>
			<td style="width:159px;">s18995</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Silencer Select ULK1-targeting siRNA (siULK1)</td>
			<td style="width:221px;">ThermoFisher/Ambion</td>
			<td style="width:159px;">s15964</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Silencer Select ULK2-targeting siRNA (siULK2)</td>
			<td style="width:221px;">ThermoFisher/Ambion</td>
			<td style="width:159px;">s18705</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Silencer Select GABARAP-targeting siRNA (siGABARAP)</td>
			<td style="width:221px;">ThermoFisher/Ambion</td>
			<td style="width:159px;">s22362</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Silencer Select GABARAPL1-targeting siRNA (siGABARAPL1)</td>
			<td style="width:221px;">ThermoFisher/Ambion</td>
			<td style="width:159px;">s24333</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Silencer Select GABARAPL2-targeting siRNA (siGABARAPL2)</td>
			<td style="width:221px;">ThermoFisher/Ambion</td>
			<td style="width:159px;">s22387</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Sodium phosphate monobasic dihydrate (NaH2PO4 &#8226; 2H2O)&#160;</td>
			<td style="width:221px;">Merck-Millipore</td>
			<td style="width:159px;">1.06580.1000</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Sodium phosphate dibasic dihydrate (Na2HPO4 &#8226; 2H2O )&#160;</td>
			<td style="width:221px;">Prolabo</td>
			<td style="width:159px;">28014.291</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Sucrose</td>
			<td style="width:221px;">VWR</td>
			<td style="width:159px;">443816T</td>
			<td>10% final concentration in water; filter through 0.45 &#181;m filter and keep in fridge for months</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Thapsigargin</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:159px;">T9033</td>
			<td>Inhibits the SERCA ER Ca2+ pump. Dissolve in DMSO.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Triton X-405&#160;</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:159px;">X405</td>
			<td style="width:703px;">1% final</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Trypan Blue stain 0.4%</td>
			<td style="width:221px;">Molecular Probes</td>
			<td style="width:159px;">T10282</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Trypsin-EDTA (0.25% w/v Trypsin)</td>
			<td style="width:221px;">Gibco</td>
			<td style="width:159px;">25200-056</td>
			<td style="width:703px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:487px;">Tween-20</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:159px;">P2287</td>
			<td>0.01% final</td>
		</tr>
	</tbody>
</table>

the lactate dehydrogenase sequestration assay &#8212; a simple and reliable method to determine bulk autophagic sequestration activity in mammalian cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Using the protocol described here, bulk autophagic sequestration activity in a number of different mammalian cell lines, including LAPC4, DU145, Huh7, PNT2A, HeLa, VCaP, H3122, Hec1A, MCF-7, T47D, U2OS, PC3, G361, mouse embryonic fibroblasts (MEFs), RPE-1, HEK293, BJ, and LNCaP cells was measured. Sequestration was assessed under basal conditions (in complete, nutrient-rich medium), or in cells acutely starved for serum and amino acids (a bona fide autophagy-inducing condition<su...

Watch this Scientific Journal Video about The Lactate Dehydrogenase Sequestration Assay â€” A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells at 

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

This method can help answer key questions in the autophagy field, such as how autophagosome formation is regulated and how various cell treatments and conditions affect bulk autophogy. The main advantage for this technique is that it measures endogenous cargo sequestration. Therefore, the method is broadly applicable and independent of artificial overexpression or introduction of cargo probes.Demonstrating the procedure, will be Paula Szalai and Morten Luhr, PhD students from my laboratory. To begin, culture adherent cells in 75 square centimeter tissue culture flasks in a humidified incubator with 5%carbon dioxide at 37 degrees celsius. Allow the cells to grow until they reach a near confluent cell layer.After this, wash the cells with three milliliters of PBS at 37 degrees celsius in pH 7.4. Remove the PBS and replace it with three milliliters of 0.25%trypsin EDTA. Incubate in a humidified incubator with 5%carbon dioxide at 37 degrees celsius, until the cells detach.Next, resuspended the cells in seven milliliters of culture medium containing 10%FBS, and transfer to a sterile 50 milliliter tube. Using a 0.5 to 20 microliter pipette tip, transfer 10 microliters of cell suspension to a microcentrifuge tube containing 10 microliters of trypan blue. Use the same pipette tip to immediately fill a counting chamber slide and count the cells in an automated cell counter.Then use culture medium containing 10%FBS to prepare suitable dilution of the cell suspension. Using aseptic techniques, seed one milliliter of the dilute cell suspension into each well of a 12 well tissue culture plate. Incubate in a humidified incubator with 5%carbon dioxide at 37 degrees celsius until the desired cell density has been reached.At the desired experimental treatments leaving one set of wells untreated in order to define the background levels of sedimentable LDH. Three to four hours before the harvest, add a saturating amount of post sequestration inhibitor Bafilomycin A1 to each well. Incubate in a humidified incubator with 5%carbon dioxide at 37 degrees celsius.At the end of the treatment period, you suction to aspirate the medium. Add 200 microliters of cell detachment solution preheated to 37 degrees celsius to each well. Incubate at 37 degrees celsius until the cells detach.Add 500 microliters of room temperature PBS at pH 7.4 containing 2%BSA to each well. Using a pipette, resuspended each well until no cell clumps are visible. After this, immediately transfer the cell suspension in each well to a separate 1.5 milliliter microcentrifuge tube on ice.Centrifuge the tubes at 400 times g and four degrees celsius for five minutes to sediment the cells. Use suction to thoroughly aspirate the supernatant, leaving the cell pellets as dry as possible. Then, add 400 microliters of solution containing 10%sucrose in ultra pure water to each tube.Using a pipette, resuspended the cell pellet to obtain a near single cell suspension. Transfer this suspension to a four millimeter electroporation cuvette. Place the cuvette in an exponential decay wave electroporator and discharge a single electric pulse at 800 volts, 25 microfarads and 400 ohms.Use a new pipette tip to transfer the cell disruptate into a 1.5 milliliter microcentrifuge tube containing 400 microliters of ice-cold phosphate buffered sucrose solution and mix briefly by pipetting. Repeat this resuspension, electro-disruption and mixing process for each sample. If performing the procedure for the first time, make sure to verify efficient and selective plasma membrane electro-disruption as described in the text.To obtain sedimented LDH fractions, remove 550 microliters from each diluted cell-disruptate solution and transfer it to its own two milliliter microcentrifuge tube containing 900 microliters of ice-cold resuspension buffer with 0.5%BSA and 0.01%Tween 20. Pipette briefly to mix. Centrifuge at 18, 000 times g and four degrees celsius for 45 minutes to produce pellets containing sedimented LDH.Using suction, thoroughly aspirate the supernatant to leave the pellets as dry as possible. Store the sedimented LDH samples at minus 80 degrees celsius until ready to analyze. For total LDH fractions, transfer 150 microliters from each of the diluted cell disruptate solutions to its own new tube.Store these in a minus 80 degrees Celsius freezer until ready to analyze. Thaw both the sedimented LDH and the total LDH samples on ice. Once thawed, add 300 microliters of ice-cold resuspension buffer containing 1.5%Triton X-405 to the total LDH samples.Rotate the samples on a roller in a cold room for 30 minutes. Next, add 750 microliters of ice-cold resuspension buffer containing 1%Triton X-405 to the sedimented LDH samples. Use a pipette to resuspended the pellets until the solution is homogenous.Centrifuge all of the samples at 18, 000 times g and four degrees celsius for five minutes to sediment undissolved cellular debris. After centrifugation is complete, put the samples on ice. Determine the levels of LDH in the supernatants using either a classic enzymatic assay as described in the text or any of the wide variety of commercially available kits.In this study, the bulk autophagic sequestration activity is monitored in a number of different mammalian cell lines. As shown here, both the basal and starvation-induced LDH sequestration and LNCaP cells are completely abolished by the pan-PI3-kinase inhibitor, 3MA. The selective PI3-kinase Class three inhibitor, SAR-405 and the ER calcium pump inhibitor, Thapsigargin.In HEK293 cells, LDH sequestration under starvation conditions is strongly reduced by both Thapsigargin and by the ULK inhibitor, MRT 67307. Moreover, starvation-induced LDH sequestration is shown to be consistently inhibited by 3MA in the other observed cell lines. Next, various ATG gene knockout MEFs are employed to test if autophagy related genes reported to be essential for autophagosome formation are required for LDH sequestration.As can be seen, starvation-induced LDH sequestration is abolished in ATG5 knockout MEFS. Moreover, it is also blunted in ATG7 and ATG9A knockout MEFs. Finally, the effects of RNAi mediated silencing of key ATG gene transcripts are determined.Transfection with an ATG9A targeting siRNA or with combined targeting of ULK1 and ULK2 is seen to strongly reduce LDH sequestration in LNCaP cells. While attempting this procedure, it's important to be accurate in all pipetting steps. Following this procedure other methods like the long-lived protein degradation assay can be performed in order to answer additional questions like were there observed alterations in sequestration activity resulting comparable changes in autophagic degradation activity.After establishing this method in mammalian cell lines, we have used it to demonstrate that bulk autophagy is independent of the LC3 protein family and instead requires GABARAPs. This highlights the importance of employing LC3 independent methods to monitor autophagy activity.

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