eoe sub articles

EoE Sub Articles

1. Cell Seeding and Treatment
<ol>
	<li>Culture adherent cells in 75 cm2&#160;tissue culture flasks in a humidified incubator with 5% CO2&#160;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.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Use Roswell Park Memorial Institute (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&#160;37 &#176;C phosphate-buffered saline (PBS), pH 7.4. Replace the PBS with 3 mL&#160;0.25% (w/v) Trypsin-ethylenediaminetetraacetate (EDTA), and incubate the flask in a humidified incubator with 5% CO2&#160;at 37 &#176;C until the cells detach (2&#8211;5 min).</li>
		<li>Resuspend the detached cells with a 7 mL&#160;culture medium containing 10% FBS. Mix a 10 &#181;L cell suspension aliquot with 10 &#181;L 0.4% Trypan Blue in a microcentrifuge tube, using a 0.5&#8211;20 &#181;L pipette tip. Use the same pipette tip to immediately fill a counting chamber slide, and count the cells in an automated cell counter.</li>
	</ol>
	</li>
	<li>Prepare a suitable dilution (see note below) of the cell suspension from step 1.1.2 using a culture medium containing 10% FBS, and seed 1&#160;mL of the diluted cell suspension in each well of a 12-well tissue culture plate (surface area ~3.8 cm2) using aseptic technique. Allow growth in a humidified incubator with 5% CO2&#160;at 37 &#176;C until the desired cell density has been reached,&#160;e.g.,&#160;60&#8211;90% confluence at harvest.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: The appropriate dilution of the cell suspension that will give the desired cell confluency at harvest will vary from cell type to cell type, as well as according to the duration and type of experimental treatments. Thus, this must be empirically assessed in each case.
	<ol>
		<li>For experiments that are to be both treated and harvested 2 days after seeding, seed 2.5 x 105&#160;LNCaP,&#160;HEK293, or MCF-7 cells, 5 x 104&#160;MEFs, 4 x 105&#160;BJ, or 1.5 x 105&#160;RPE-1&#160;cells in each well of the 12-well plate.</li>
		<li>For cells that adhere loosely, coat the plates with the type of coating recommended for the cell type in question. For LNCaP (and HEK293) cells use plates coated with poly-D-lysine (PDL).</li>
		<li>To that end, add 500 &#181;L PDL at 2.5 &#181;g/mL in sterile H2O to each well, and incubate the plates in a sterile environment for 30 min at room temperature (20&#8211;25 &#176;C). Remove the PDL with suction, and wash each well briefly with 1 mL sterile H2O.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: Generally, step 1.2.1 is done without any experimental treatments. However, if performing RNAi, it may be convenient to start a reverse transfection with the seeding.</li>
	</ol>
	</li>
	<li>Perform experimental treatments in duplicate or triplicate wells per condition.
	<ol>
		<li>For example, treat the cells with 50 nM of the mTOR-inhibitor Torin1, which generally is an efficient inducer of autophagic sequestration, or subject the cells to acute serum- and amino acid starvation by washing the cells with 1 mL of amino acid-free Earle&#39;s Balanced Salt Solution (EBSS) medium, and subsequently incubate the cells in 1 mL of EBSS in a humidified incubator with 5% carbon dioxide (CO2) at 37 &#176;C.</li>
		<li>Leave one set of wells untreated in order to define background levels of sedimentable lactate dehydrogenase (LDH).</li>
		<li>Add a saturating amount of the post-sequestration inhibitor bafilomycin A1 (Baf)&#160;in the absence or presence of the experimental treatments, 3&#8211;4 h before cell harvest. Incubate the cells in a humidified incubator with 5% CO2&#160;at 37 &#176;C.
		<ol>
			<li>Use 100 nM Baf for LNCaP, HEK293, BJ, MCF-7, and RPE-1 cells, and 10 nM Baf for MEFs.</li>
			<li>For experimental treatments that&#160;have a duration of only 3&#8211;4 h (like those exemplified in step 1.3.1 typically have), add Baf simultaneously with the treatments. For longer experimental treatments, wait until 3-4 h before harvest, and add 2 &#181;L&#160;of a 500x concentrated Baf stock directly into the medium.</li>
			<li>Mix by agitating the plate immediately after the addition of Baf. At this point, it is also recommendable to add macroautophagic sequestration inhibitors as controls,&#160;e.g.,&#160;10 mM of the pan-phosphoinositide 3-kinase (PI3K) inhibitor 3-methyl adenine (3MA), or 10 &#181;M of the selective PI3K class III inhibitor SAR-405.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Cell Harvest and Preparation for Electrodisruption
<ol>
	<li>At the end of the treatment period, aspirate the medium with suction and add 200 &#181;L cell detachment solution (pre-heated to 37 &#176;C) to each well. Incubate at 37 &#176;C until the cells detach (typically around 5 min).&#160;&#160;&#160;&#160; 
	NOTE: Whereas 0.25% (w/v) Trypsin-EDTA may be used instead of the cell detachment solution, the latter contains DNase, which helps reduce the viscosity of the detached cells. As long as the medium is thoroughly aspirated, it is not necessary to wash the cells before the addition of Trypsin-EDTA or cell detachment solution.</li>
	<li>Add 500 &#181;L room temperature (20-25 &#176;C) PBS, pH 7.4, containing 2% (w/v) bovine serum albumin (BSA) to each well, and resuspend with the pipette until no cell clumps are visible. Immediately transfer the cell suspension to 1.5 mL microcentrifuge tubes on ice.&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Unless otherwise stated, perform all subsequent steps on ice.</li>
	<li>Sediment the cells by centrifugation at 400 x&#160;g&#160;for 5 min at 4 &#176;C.</li>
	<li>Thoroughly aspirate the supernatant (with suction) to leave the cell pellets as dry as possible.</li>
	<li>Add 400 &#181;L 10% (w/v) sucrose (in ultrapure H2O) to each tube.</li>
</ol>
3. Plasma Membrane Electrodisruption and Separation of Sedimentable- and Total-cell Fractions
<ol>
	<li>Resuspend the cell pellet with a pipette to obtain a near single-cell suspension, and transfer it to a 4 mm electroporation cuvette.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Pipetting up and down ~10&#8211;15 times, using a 100&#8211;1,000 &#181;L pipette tip, is usually sufficient.</li>
	<li>Place the cuvette in an exponential decay wave electroporator, and discharge a single electric pulse at 800 V, 25 &#181;F, and 400 &#8486;; these settings produce a pulse of ~8 ms duration.</li>
	<li>Use a new pipette tip to transfer the cell disruptate to a 1.5 mL microcentrifuge tube containing 400 &#181;L ice-cold phosphate-buffered sucrose solution (100 mM sodium monophosphate, 2 mM dithiothreitol (DTT), 2 mM EDTA, and 1.75% sucrose, pH 7.5), and mix briefly by pipetting.
	<ol>
		<li>Optional: To verify efficient plasma membrane electro-disruption, mix 10 &#181;L of the diluted cell disruptate from step 3.3 with 10 &#181;L 0.4% Trypan Blue in a 1.5 mL microcentrifuge tube. Transfer to a counting chamber and verify that the percentage of Trypan Blue positive cells is &#62;99%.
		<ol>
			<li>Leave the sample in the counting chamber for 30 min at room temperature (20&#8211;25 &#176;C), and verify that the percentage of Trypan Blue positive cells has remained &#62;99%.</li>
		</ol>
		</li>
		<li>Optional: To verify that the electro-disruption has not been too harsh, that is, it has not disrupted intracellular organelles, perform steps 1.1&#8211;3.3 as described above, but use a larger starting material (a well from a 6-well plate with an ~80% confluent cell layer), and use 150 &#181;L 10% sucrose in step 2.5 and 150 &#181;L phosphate-buffered sucrose solution without DTT in step 3.3.
		<ol>
			<li>Use a pipette to carefully layer 200 &#181;L of the diluted cell disruptate solution on top of a 1.2 mL density cushion of phosphate-buffered 8% (w/v) density gradient medium (e.g, 8% Nycodenz, 50 mM sodium phosphate, 2.2% sucrose, 1 mM EDTA) in a 2 mL centrifuge tube. Centrifuge at 20,000 x&#160;g&#160;for 45 min at 4 &#176;C in a microcentrifuge with soft-mode function (for gentle acceleration and deceleration), and carefully put the tubes on ice.</li>
			<li>Carefully remove 60 &#181;L of the ~200 &#181;L top fraction, making sure not to pick up any density gradient medium solution, and transfer to a fresh microcentrifuge tube.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
			NOTE: This should contain cytosol of exceptional purity, termed &#34;cell sap&#34;.</li>
			<li>Test the purity of the fraction obtained in the above step, by performing western blot analyses of organelle-contained proteins, using standard techniques and 4&#8211;20% gradient gels.</li>
			<li>Perform for example immunoblotting for cathepsin B, cytochrome c, and protein disulfide isomerase, to verify that the electric shock in step 3.2 has not disrupted lysosomes, mitochondria, or endoplasmic reticulum, respectively, and immunoblot for LDH to verify the presence of a cytosolic protein in the cell sap.</li>
			<li>In parallel, perform immunoblotting on protein extracts made from the total cell disruptate solution&#160;to confirm that the antibodies used can detect the organelle-contained proteins that are being assessed.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Repeat steps 3.1&#8211;3.3 for each sample.</li>
	<li>Remove 550 &#181;L from each diluted cell disruptate solution (obtained at step 3.3) to 2 mL microcentrifuge tubes containing 900 &#181;L ice-cold resuspension buffer (50 mM sodium monophosphate, 1 mM DTT, 1 mM EDTA, and 5.9% sucrose, pH 7.5) supplemented with 0.5% BSA and 0.01% Tween-20, and mix briefly by pipetting.</li>
	<li>Centrifuge at 18,000 x&#160;g&#160;for 45 min at 4 &#176;C to produce pellets containing &#34;sedimented LDH&#34;. Thoroughly aspirate the supernatant (with suction) to leave the pellets as dry as possible. Place the samples in a -80 &#176;C freezer.</li>
	<li>Transfer 150 &#181;L from each diluted cell disruptate solution (obtained at step 3.3) to new tubes, and place the samples in a -80 &#176;C freezer. Use these samples to determine the &#34;total LDH&#34; levels in the cells.&#160;&#160;&#160; 
	NOTE: At this point, the experiment can be paused for as long as desired.</li>
</ol>
4. LDH Extraction and Measurement of LDH Enzymatic Activity
<ol>
	<li>Thaw the &#34;sedimented LDH&#34; (from step 3.6) and &#34;total LDH&#34; samples (from step 3.7) on ice.</li>
	<li>Add 300 &#181;L of ice-cold resuspension buffer containing 1.5% Triton X-405 to the &#34;total LDH&#34; samples (yielding a final Triton X-405 concentration of 1%). Rotate the samples on a roller in a cold room (4&#8211;8 &#176;C) for 30 min.</li>
	<li>Add 750 &#181;L of ice-cold resuspension buffer with 1% Triton X-405 to the &#34;sedimented LDH&#34; samples, and resuspend the pellets with a pipette until a homogenous solution is reached.</li>
	<li>Centrifuge the samples from steps 4.2 and 4.3 at 18,000 x&#160;g&#160;for 5 min at 4 &#176;C&#160;to sediment undissolved cellular debris.</li>
	<li>Mix 4 parts of cold 65 mM imidazole (pH 7.5)/0.75 mM pyruvate with one part of cold 65 mM imidazole (pH 7.5)/1.8 mM NADH to obtain a working solution that is stable for at least three weeks at 4 &#176;C.</li>
	<li>Mix 3&#8211;30 &#181;L of the supernatants from step 4.4 with 200 &#181;L of the step 4.5 working solution.</li>
	<li>Determine the amount of LDH by measuring LDH enzymatic activity as the decline in nicotinamide adenine dinucleotide (reduced form) (NADH) absorbance at 340 nm at 37 &#176;C compared to a standard with a known LDH concentration. Perform absorbance measurements&#160;until the reaction has approached completion,&#160;i.e.&#160;until the absorbance at 340 nm no longer changes with time.&#160; 
	NOTE: This is the classical biochemical method to measure LDH activity. Although the current protocol performs the reaction at 37 &#176;C, it can also be performed at room temperature (20&#8211;25 &#176;C), which is advisable if doing manual spectrophotometry. The current protocol uses a robotic multi-analyzer instrument, which in an automated fashion mixes samples with working solution in a 96-well plate, and measures the absorbance at 340 nm at 37 &#176;C every 20 s for 3 min. Thereafter, the instrument software calculates the concentration of LDH, expressed as Units (U)/L, by comparing the slope of the absorbance measurements over time compared to a standard curve obtained through&#160;calibration with a standard of known LDH concentration. The linear range of detection by this approach is 30&#8211;1,500 U/L. As an alternative, a wide variety of commercially available kits to measure LDH exist. Some of them are based on coupling the enzymatic reaction to the generation of colorimetric or fluorescent products, enabling detection by other means than UV spectrophotometry, and with other linear ranges of detection.</li>
</ol>

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

quantifying bulk autophagic sequestration activity in mammalian cells

Source: Luhr, M. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57971">The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells.</a> J. Vis. Exp. (2018)
The video demonstrates an assay for assessing bulk autophagic sequestration activity in mammalian cells. It utilizes electroporation to release cytosolic proteins, including lactate dehydrogenase (LDH). The sequestered LDH and total cellular LDH fractions are determined to assess LDH sequestration, providing insights into the overall bulk sequestration activity.

Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells

1. Cell Seeding and Treatment

	Culture adherent cells in 75 cm2&#160;tissue culture flasks in a humidified incubator with 5% CO2&#160;at 37 &#176;C, ...

Bulk autophagy involves sequestering autophagic cargo and soluble cytosolic proteins, including lactate dehydrogenase or LDH, within autophagosomes for degradation.
To assess LDH sequestration, reflecting bulk autophagic activity, take an electroporation cuvette with nutrient-starved cells treated with inhibitors to block autophagic-lysosomal degradation.
Perform electroporation to selectively disrupt the cell membrane and release soluble proteins.
Mix the cell disruptate with buffered sucrose solution to preserve cellular components.
Transfer the required diluted disruptate solution to a buffer-containing tube. Centrifuge to separate non-sequestered LDH from autophagosome-sequestered LDH.
Transfer the remaining diluted disruptate solution, containing the total cellular LDH fraction, to another tube.
Add a non-ionic surfactant-containing buffer to both tubes to extract sequestered and total LDH fractions.
Centrifuge and transfer LDH-containing supernatants to buffer-containing tubes with pyruvate and NADH.
LDH converts pyruvate to lactate with NADH oxidation, decreasing NADH levels correlating with LDH concentration.
Quantify LDH in sedimented and total cellular fractions to determine LDH sequestration.

quantifying-bulk-autophagic-sequestration-activity-in-mammalian-cells

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

38 Views. Źródło: Luhr, M. et al., Test sekwestracji dehydrogenazy mleczanowej — prosta i niezawodna metoda określania masowej aktywności sekwestracji autofagicznej w komórkach ssaków. J. Vis. Exp. (2018) Film demonstruje test do oceny masowej aktywności sekwestracji autofagicznej w komórkach ssaków. Wykorzystuje elektroporację do uwalniania białek cytozolowych, w tym dehydrogenazy mleczanowej (LDH). Sekwestrowane LDH i całkowite komórkowe frakcje LDH są oznaczane w celu oceny sekwestrac...

Video: Kwantyfikacja masowej aktywności sekwestracji autofagicznej w komórkach ssaków - Concept

A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have demonstrated an altered population of circulating and decidual NK cells in women who suffer from adverse pregnancy complications such as recurrent miscarriage and preeclampsia. Studies from our group have shown that hypertension in pregnancy is associated with an increased population of activated NK cells in the placenta based on the expression of surface activation markers. This manuscript provides a detailed protocol to assess the cytotoxic function of NK cells isolated from placentas in a preeclampsia-like animal model of surgically induced placental ischemia. The following steps are described in detail: generation of single cell suspension, NK cell isolation, ex vivo stimulation, effector:target cell co-culture, and the cytotoxicity assay.

Here, we provide a detailed methodology to isolate and assess cytotoxic function of natural killer cells from placentas by a colorimetric plate assay. The reduced uterine perfusion pressure rat model of placental ischemia was chosen to demonstrate the antibody-mediated isolation and assessment of the cytotoxic function of natural killer cells.

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have ...

JoVE Journal

Immunology and Infection

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

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.

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 ...

Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells

Transkrypcja

Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells