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&#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 at 37 &#176;C until the cells detach (2&#8211;5 min).</li>
		<li>Resuspend the detached cells with 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 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 at 37 &#176;C until the desired cell density has been reached, e.g., 60&#8211;90% confluence at harvest. 
	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 LNCaP,&#160;HEK293, or MCF-7 cells, 5 x 104 MEFs, 4 x 105 BJ, or 1.5 x 105 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. 
		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 seeding9.</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% CO2 at 37 &#176;C.</li>
		<li>Leave one set of wells untreated in order to define background levels of sedimentable LDH.</li>
		<li>Add a saturating amount of the post-sequestration inhibitor bafilomycin A1 (Baf)3,13,16,18 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 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, e.g., 10 mM of the pan-phosphoinositide 3-kinase (PI3K) inhibitor 3-methyl adenine (3MA)19, or 10 &#181;M of the selective PI3K class III inhibitor SAR-40520.</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). 
	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 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. 
	NOTE: Unless otherwise stated, perform all subsequent steps on ice.</li>
	<li>Sediment the cells by centrifugation at 400 x g 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. 
	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 electrodisruption17, 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 electrodisruption 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 g 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. 
			NOTE: This should contain cytosol of exceptional purity, termed &#34;cell sap&#34;21.</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 gels16.</li>
			<li>Perform for example immunoblotting for cathepsin B21, cytochrome c, and protein disulphide 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 presence of a cytosolic protein in the cell sap.</li>
			<li>In parallel, perform immunoblotting on protein extracts made from the total cell disruptate solution16 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 g 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. 
	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 step 4.2 and 4.3 at 18,000 x g 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, i.e. until the absorbance at 340 nm no longer changes with time. 
	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 multianalyzer 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>
5. Calculation of LDH Sequestration
<ol>
	<li>Calculate the percentage of sedimented LDH to total LDH for each sample, taking the dilutions and sampling into account: 
	Sedimented LDH (%) = <img alt="Equation 1" src="/files/ftp_upload/57971/57971eq1.jpg" /> 
	NOTE: During steps 3.1&#8211;3.3 approximately 50 &#181;L is lost due to transfer into and out of the electroporation cuvette. Thus, calculate from having a total of 750 &#181;L (instead of 800 &#181;L) of diluted cell disruptate in step 3.3.</li>
	<li>Subtract the percentage of sedimented LDH obtained in the samples from untreated cells (step 1.3.2) from the percentage of sedimented LDH obtained in samples from experimentally treated cells, and divide by the treatment time with Baf to obtain the percentage of sequestered LDH per hour in the sampling period: 
	Sequestered LDH (%/h) = <img alt="Equation 2" src="/files/ftp_upload/57971/57971eq2.jpg" /></li>
</ol>

The authors have no conflict of interest.

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:1569px;" width="1567">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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 Ã¢â‚¬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 ...

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18.0K Views.  University of Oslo. 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.

Video: The Lactate Dehydrogenase Sequestration Assay — A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...