We thank the members of the Division of Genome Science and Laboratory for Systems Biology and Medicine, RCAST, The University of Tokyo. We especially thank Dr. H. Aburatani, Dr. T. Kodama, (LSBM, RCAST, The University of Tokyo), Dr. K. Tomizuka, Dr. T. Yoshida, and Dr. A. 
Kunisato (Kyowa Hakko Kirin Co., Ltd.) for helpful discussions and support. This work was partly supported by Grant-in-Aid for Young Scientist (A) (26710005, T.O.), Grant-in-Aid for Scientific Research on Innovative Areas (26116711 and 16H01567, T.O.), and Grant-in-Aid for Challenging 
Exploratory Research (16K14605, T.O.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Takeda Science Foundation (T.O.), the Kobayashi Foundation of Cancer Research (T.O.), and the Project for Cancer Research and Therapeutic Evolution (P-CREATE) and the Practical Research for Innovative Cancer Control from Japan Agency for Medical Research and Development, AMED (T.O.).

1. Preparation of Acidic Culture Medium
<ol>
	<li>Preparation of control (pH 7.4) and low-pH (pH 6.8) culture medium
	<ol>
		<li>Dissolve 4.75 g of DMEM powder without L-glutamine in 500 mL of water.</li>
		<li>Prepare 0.33 M NaHCO3 solution in a pressure-tight bottle. 
		Caution: It is important to use a pressure tight bottle since NaHCO3 emits CO2 gas when heated and thermally decomposed.</li>
		<li>Autoclave the medium and solution. Allow these buffers to cool to 25 &#176;C.</li>
		<li>Add 5 mL of 200 mM L-glutamine, 5 mL of penicillin-streptomycin, and 50 mL of fetal bovine serum to 500 mL of DMEM without bicarbonate.</li>
		<li>For control (pH 7.4) medium, add 2.4 mL of 0.33 M NaHCO3 solution per 100 mL of DMEM without bicarbonate. For low-pH (pH 6.8) medium, add 0.6 mL of 0.33 M NaHCO3 solution per 100 mL of DMEM without bicarbonate.</li>
		<li>Check the medium pH after 24 h of incubation at 37 &#176;C under 5% CO2. 
		NOTE: The amount of NaHCO3 solution needs to be adjusted depending on the medium pH, which can differ depending on the CO2 content of the incubator and air pressure. Ideally, for the first time, it is better to measure the medium pH with various bicarbonate concentrations to determine the appropriate amount of NaHCO3 solution to add to DMEM.
		<ol>
			<li>Collect culture medium immediately after removing the culture dish from the CO2 incubator and measure the pH using a pH meter. Keep the medium temperature at 37 &#176;C using a water bath if necessary. Measure the medium pH three times independently. 
			NOTE: NaHCO3 solution should be stored at 4 &#176;C and each media should be freshly prepared. Commercially available products which can be used in this study are listed in Table of Materials.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparing lactate-induced or HCl-induced acidic culture medium
	<ol>
		<li>Add 74 &#181;L of lactate solution or 125 &#181;L of 1 M HCl to 100 mL of control medium (prepared in step 1.1.5).</li>
		<li>Check the medium pH after 24 h of incubation at 37 &#176;C under 5% CO2. 
		NOTE: The amount of lactate or HCl solution also needs to be adjusted according to the medium pH, which can differ depending on the CO2 content in the incubator and air pressure. Ideally, it is better to measure the medium pH with serial lactate or HCl concentrations to determine the appropriate amount for adjusting to the desired pH of the medium.</li>
	</ol>
	</li>
</ol>
2. Harvesting and Treating Cells
<ol>
	<li>Start cultivating cells at 37 &#176;C under 5% CO2 at least 1 week before performing experiments. 
	NOTE: The cell lines used in this study are listed in Table of Materials.</li>
	<li>Passage cells when they reach 70 - 90% confluency. Do not allow the cells to become confluent. Change the medium every 2 - 3 days during daily cultivation.</li>
	<li>Wash the cells by adding 5 mL PBS. Aspirate the PBS and add detaching enzyme such as 1 mL Trypsin.</li>
	<li>Incubate at 37 &#176;C until the cells are rounded and start to detach.</li>
	<li>Add 5 mL of culture medium to the detached cells and deactivate enzyme. Transfer the cell suspension to a 15 mL tube and centrifuge for 3 min at 300 x g. Aspirate the supernatant and re-suspend the cells with culture medium.</li>
	<li>Count the viable cells using a hemocytometer and Trypan-Blue.</li>
	<li>Seed 5.0 x 105 cells/well of a 10-cm dish in 10 mL of medium. Incubate the cells for 24 h at 37 &#176;C under 5% CO2.</li>
	<li>Aspirate the culture medium, wash the cells with 5 mL PBS, and add 10 mL of the acidic culture medium prepared in Section 1. Incubate the cells for 24 h at 37 &#176;C under 5% CO2.</li>
</ol>
3. RNA Isolation
<ol>
	<li>Aspirate culture medium and wash cells with 5 mL PBS. Disrupt the cells by adding RNA extraction reagent (see Tables of Materials).</li>
	<li>Scrape the dish briefly, then remove the RNA extraction reagent with a pipette and deposit the RNA extraction reagent/cell lysate into a 1.5 mL tube. Leave at room temperature for 5 min.</li>
	<li>Add 250 &#181;L chloroform and shake the tube vigorously for about 15 s. Leave at room temperature for 5 min and centrifuge at &#8805;8,000 x g for 5 min.</li>
	<li>Carefully remove the aqueous phase using a pipette. Leave behind some of the aqueous phase. Place in another 1.5 mL tube.</li>
	<li>Add 550 &#181;L isopropanol to the aqueous phase and mix gently. Leave at room temperature for 5 min. Centrifuge at maximal speed (&#8805;20,000 x g) for 20 min at 4 &#176;C.</li>
	<li>Pour off the isopropanol and add 1 mL 75% ethanol in DEPC treated water. Mix gently. Centrifuge at 8,000 x g for 5 min.</li>
	<li>Pipette off the ethanol and let the pellets air-dry.</li>
	<li>Add approximately 15 - 25 &#181;L (depending on the yield) of DEPC treated water to the RNA pellet. Mix thoroughly.</li>
	<li>Measure the concentration of isolated RNA by measuring the OD at 260 nm using a spectrophotometer.</li>
</ol>
4. cDNA Synthesis
<ol>
	<li>Combine the following components in a PCR reaction tube: up to 5 &#181;g total RNA, 1 &#181;L Oligo dT (50 &#181;M) or Random Primer Mix (60 &#181;M), 1 &#181;L 10 mM dNTP, and nuclease-free water to a total volume of 14 &#181;L.</li>
	<li>Mix and briefly centrifuge the components using a small benchtop centrifuge (~5 s). Denature the sample RNA/primer for 5 min at 65 &#176;C. Spin briefly (~5 s) using a small benchtop centrifuge and put the sample promptly on ice for at least 1 min.</li>
	<li>Add the following components: 4 &#181;L 5x&#160;Reverse Transcription buffer, 1 &#181;L Reverse Transcriptase (200 U/&#181;L), 1 &#181;L RNase Inhibitor (40 U/&#181;L). 
	NOTE: Examples of commercially available kits are shown in Table of Materials</li>
	<li>Cap the tube, mix, and then briefly centrifuge the contents using a small benchtop centrifuge (~5 s). Incubate the combined reaction mixture at 50 - 55 &#176;C for 10 min.</li>
	<li>Inactivate the reaction by incubating at 80 &#176;C for 10 min.</li>
</ol>
5. Measurement of Acid-responsive Gene Expression Using Real-time PCR
<ol>
	<li>Combine the following components in a 384-well reaction plate: 1 &#181;L Template cDNA, 0.75 &#181;L 10X N&#39;,N&#39;-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine, 0.3 &#181;L 50 &#181;M forward primer, 0.3 &#181;L 50 &#181;M reverse primer, 1 &#181;L Taq polymerase, 2.5 &#181;L 10x&#160;Taq polymerase buffer, 2.5 &#181;L 20 mM dNTP, 16.65 &#181;L water. 
	NOTE: Example of commercially available kits are shown in Table of Materials. A list of primers is shown in Table 1. A 96-well reaction plate can be also used in this step. Other probes (e.g., Taqman probe) can also be used.</li>
	<li>Set up the experiment and the following PCR program on a Real-time PCR System: 50 &#176;C for 2 min, 1 cycle; 95 &#176;C for 10 min, 1 cycle; 95 &#176;C for 15 s and 60 &#176;C for 1 min, 40 cycles; 95 &#176;C for 15 s, 1 cycle; 60 &#176;C for 15 s, 1 cycle; 95 &#176;C for 15 s, 1 cycle.</li>
	<li>Place the plate in the qPCR instrument. Start the program. 
	NOTE: Upregulation of the pH responsive genes MSMO1, INSIG1, and IDI1 can be measured by protein levels.</li>
</ol>

<ol>
	<li>Cairns, R. A., Harris, I. S., Mak, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+cell+metabolism.">Regulation of cancer cell metabolism.</a> Nature reviews. Cancer. 11 (2), 85-95 (2011).</li><li>Chang, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Competition+in+the+Tumor+Microenvironment+Is+a+Driver+of+Cancer+Progression.">Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression.</a> Cell. 162 (6), 1229-1241 (2015).</li><li>Hsu, P. P., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+metabolism:+Warburg+and+beyond.">Cancer cell metabolism: Warburg and beyond.</a> Cell. 134 (5), 703-707 (2008).</li><li>Gerweck, L. E., Seetharaman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pH+gradient+in+tumor+versus+normal+tissue:+potential+exploitation+for+the+treatment+of+cancer.">Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer.</a> Cancer research. 56 (6), 1194-1198 (1996).</li><li>Webb, B. A., Chimenti, M., Jacobson, M. P., Barber, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulated+pH:+a+perfect+storm+for+cancer+progression.">Dysregulated pH: a perfect storm for cancer progression.</a> Nature Rev. Cancer. 11 (9), 671-677 (2011).</li><li>Gatenby, R. A., Gillies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+cancers+have+high+aerobic+glycolysis?.">Why do cancers have high aerobic glycolysis?.</a> Nature reviews. Cancer. 4 (11), 891-899 (2004).</li><li>McBrian, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+regulates+intracellular+pH.">Histone acetylation regulates intracellular pH.</a> Mol Cell. 49 (2), 310-321 (2013).</li><li>Damaghi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+acidosis+in+the+tumour+microenvironment+selects+for+overexpression+of+LAMP2+in+the+plasma+membrane.">Chronic acidosis in the tumour microenvironment selects for overexpression of LAMP2 in the plasma membrane.</a> Nat Commun. 6, 8752 (2015).</li><li>Corbet, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidosis+Drives+the+Reprogramming+of+Fatty+Acid+Metabolism+in+Cancer+Cells+through+Changes+in+Mitochondrial+and+Histone+Acetylation.">Acidosis Drives the Reprogramming of Fatty Acid Metabolism in Cancer Cells through Changes in Mitochondrial and Histone Acetylation.</a> Cell Metab. 24 (2), 311-323 (2016).</li><li>Chen, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+analysis+of+lactic+acidosis+and+acidosis+response+in+human+cancers.">The genomic analysis of lactic acidosis and acidosis response in human cancers.</a> PLoS genetics. 4 (12), e1000293 (2008).</li><li>Kondo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Acidic+pH+Activates+the+Sterol+Regulatory+Element-Binding+Protein+2+to+Promote+Tumor+Progression.">Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.</a> Cell Rep. 18 (9), 2228-2242 (2017).</li></ol>

The author Ayano Kondo is an employee of Kyowa Hakko Kirin Co., Ltd.

Here, we described a simple acidic pH culture system and its evaluation process. The combination of three methods of medium acidification, i.e., reduced bicarbonate concentration, lactate addition, and HCl addition, enabled us to investigate the pH-response mechanism thoroughly and to compare the cellular response to other tumor microenvironmental conditions such as hypoxia or nutrient starvation.
The key of this method is to determine the appropriate concentrations of bicarbonate, la...

The tumor microenvironment plays critical roles in tumor progression and cancer cell metabolism1,2,3. Cancer cells are often exposed to conditions such as hypoxia, nutrient deprivation, and acidic extracellular pH (pHe). However, the role of pHe in tumor progression has not been clarified as extensively as in hypoxia or nutrient starvation. The pHe in the tumor tissue can become acidic, reaching approximately pH 6.84,5. The acidic pH arises from aerobic and anaerobic glycolytic excretion of protons (H+) and lactate by proliferating cancer cells5,6.
Recent studies revealed that acidic pHe-induced histone deacetylation, fatty acid oxidation, and exocytosis of acidic lysosomes for adaption to the severe acidic environment7,8,9,10. However, the mechanisms through which extracellular acidification affects cancer behavior and the identity of key regulators in the acidic pH tumor microenvironment have not been fully determined. Furthermore, several reports described various acidic pH media using unclear concentrations of bicarbonate, Tris, PIPES, and HEPES buffer or lactate and HCl, but there are few reports to demonstrate the stability of the adjusted medium nor comprehensive comparison of several distinct acidic culture media7,8,9,10
To elucidate the key regulators and metabolic changes in cancer cells in the context of extracellular acidification, we established a simple in vitro culture model to maintain an acidic pHe and examined the role of pHe in cancer cells11. Using this method, we maintained an acidic culture medium with a pHe of 6.8 at 37 &#176;C under 5% CO2, using reduced bicarbonate concentrations in the culture medium. pH 7.4 was used as normal and control medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h during culture of PANC-1 and AsPC-1 pancreatic cancer cells. Because enhanced glycolysis accelerates excretion of not only protons but also lactate7,8,9, we also established a culture method mimicking lactate-induced acidosis by adding lactate rather than reducing the bicarbonate concentration. Additionally, HCl-induced acidic pHe in the medium allows us to exclude the possibility that cellular responses to acidic pH culture medium are not due to a reduced concentration of bicarbonate. Moreover, using various media with a pH of 6.4 to 7.4 with different bicarbonate concentration, we can assess the extent of pHe effects on cellular responses.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium&#8220;Nissui&#8221; 2</td>
			<td>Nissui</td>
			<td>05919</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher&#160;</td>
			<td>10438</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Wako</td>
			<td>191-01305</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine solution</td>
			<td>Thermo Fisher&#160;</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Mixed Solution(Stabilized)</td>
			<td>Nacalai</td>
			<td>09367-34</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5g/l-Tripsin/0.53mmol/l-EDTA Solution, with Phenol Red</td>
			<td>Nacalai</td>
			<td>32778-05</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5%-Trypan Blue Stain Solution</td>
			<td>Nacalai</td>
			<td>29853-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactic acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>252476</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>H9892</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Plus Mini Kit</td>
			<td>QIAGEN</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV First-Strand Synthesis System</td>
			<td>Thermo Fisher&#160;</td>
			<td>18091</td>
			<td></td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR Master Mix&#160;</td>
			<td>Applied Biosystems&#160;</td>
			<td>4368702&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PANC-1</td>
			<td>ATCC</td>
			<td>CRL-1469</td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1</td>
			<td>ATCC</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
		<tr>
			<td>KMS-6</td>
			<td>JCRB Cell Bank</td>
			<td>JCRB0432</td>
			<td></td>
		</tr>
		<tr>
			<td>TIG</td>
			<td>JCRB Cell Bank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRC9</td>
			<td>ATCC</td>
			<td>CCL-212</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>ATCC</td>
			<td>CRL-1730</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One Microvolume UV-Vis Spectrophotometer with Wi-Fi</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 5 Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of an extracellular acidic ph culture system

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

To determine the appropriate bicarbonate concentration, we prepared DMEM at range of 0 - 8 mM NaHCO3 (final concentration in the culture medium) and succeeded in preparing culture media with pH ranging from 6.4 - 7.4 (Figure 1). We prepared DMEM with 8 mM NaHCO3 (pH 7.4) as a control medium, and 2 mM NaHCO3 (pH 6.8) as a medium with acidic pH according to a previous report stating that the extracellular pH reaches pH 6.8 for s...

Watch this Scientific Journal Video about Establishment of an Extracellular Acidic pH Culture System at JoVE.com

Establishment of an Extracellular Acidic pH Culture System

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

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14.5K Views.  Kyowa Hakko Kirin Co., Ltd. The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Video: Establishment of an Extracellular Acidic pH Culture System

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

Enhanced Bacterial Extracellular Vesicles Isolation for Microbiota Multi-Omics

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

Author Spotlight: Accelerating Research on Bacterial Extracellular Vesicles Separation and Heterogeneity 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.