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>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium&amp;ldquo;Nissui&amp;rdquo; 2</td><td>Nissui</td><td>05919</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher</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</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</td><td>18091</td><td></td></tr><tr><td>Power SYBR Green PCR Master Mix&amp;nbsp;</td><td>Applied Biosystems</td><td>4368702&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Cell lines&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</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 ...

establishment-of-an-extracellular-acidic-ph-culture-system

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Biology

Unit 1

Fluid, Electrolyte, and Acid-Base Balance

SCIENTISTS IN ACTION

14.6K 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

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

In vitro Monitoring of Extracellular pH in Real-Time

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can accumulate and contribute to chronic inflammatory conditions, including the intestinal diseases ulcerative colitis (UC) and Crohn's Disease (CD). The pH in the distal colon in individuals with active UC can range between a pH of 5 and 6, whereas healthy individuals maintain colonic pH in the range of 6.8-7.4. Extracellular pH has been shown to influence both intestinal epithelial cells and the infiltrating immune cells. More specifically, extracellular acidosis significantly impacts PMN. At pH below 6.5, there are increases in the production of H2O2, inhibition of apoptosis, and increases in the functional lifespan of PMN. Given the significant presence of PMN and extracellular acidification at sites of inflammation, we developed a novel model that allows for the monitoring of extracellular pH during PMN transepithelial migration in real time. Here, we describe this model and how it can be utilized to measure both the apical and basal pH during PMN trafficking. This model can be utilized to monitor extracellular pH under a wide range of conditions; including, hypoxia, PMN transepithelial migration, and for extended periods of time.

In vitro Monitoring of Extracellular pH in Real-Time

This article represents a useful in vitro assay to measure changes in extracellular pH during neutrophil (PMN) transepithelial migration (TEM)

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to ...

Chronoamperometry Analysis of EET in Lactiplantibacillus plantarum with DHNA

Characterizing Mediated Extracellular Electron Transfer in Lactic Acid Bacteria with a Three-Electrode, Two-Chamber Bioelectrochemical System

Many bacteria perform extracellular electron transfer (EET), whereby electrons are transferred from the cell to an extracellular terminal electron acceptor. This electron acceptor can be an electrode and electrons can be delivered indirectly via a redox-active mediator molecule. Here, we present a protocol to study mediated EET in Lactiplantibacillus plantarum, a probiotic lactic acid bacterium widely used in the food industry, using a bioelectrochemical system. We detail how to assemble a three-electrode, two-chambered bioelectrochemical system and provide guidance on characterizing EET in the presence of a soluble mediator using chronoamperometry and cyclic voltammetry techniques. We use representative data from 1,4-dihydroxy-2-naphthoic acid (DHNA)-mediated EET experiments with L. plantarum to demonstrate data analysis and interpretation. The techniques described in this protocol can open new opportunities for electro-fermentation and bioelectrocatalysis. Recent applications of this electrochemical technique with L. plantarum demonstrated an acceleration of metabolic flux towards producing fermentation end-products, which are critical flavor components in food fermentation. As such, this system has the potential to be further developed to alter flavors in food production or produce valuable chemicals.

Here we present a protocol for characterizing mediated extracellular electron transfer (EET) in lactic acid bacteria using a three-electrode, two-chamber bioelectrochemical system. We illustrate this method with Lactiplantibacillus plantarum and the redox mediator 1,4-dihydroxy-2-naphthoic acid and provide a thorough description of the electrochemical techniques used to evaluate mediated EET.

Many bacteria perform extracellular electron transfer (EET), whereby electrons are transferred from the cell to an extracellular terminal electron ...

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...

Bioengineering

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment. Despite vast differences in the physiology between mammals and fish, primary cell culture protocols from fish are often based on mammalian culture conditions, often with only minor modifications. The environmental differences affect not only body temperature, but also blood serum parameters such as osmolality, pH, and pH buffer capacity. As cell culture media and similar working solutions are meant to mimic characteristics of the extracellular fluid and/or blood serum to which a cell is adapted, it is crucial that these parameters are adjusted specifically to the animal in question.
The current protocol describes optimized primary culture conditions for medaka (Oryzias latipes). The protocol provides detailed steps on how to isolate and maintain healthy dissociated pituitary cells for more than one week and includes the following steps: 1. the adjustment of the osmolality to the values found in medaka blood plasma, 2. the adjustment of the incubation temperature to normal medaka temperature (here in the aquarium facility), and 3. the adjustment of the pH and bicarbonate buffer to values comparable to other fish species living at similar temperatures. The results presented using the described protocol promote physiologically meaningful results for medaka and can be used as a reference guide by scientists making primary cell cultures from other non-mammalian species.

Here we describe a protocol to prepare and maintain primary pituitary cell cultures from medaka (Oryzias latipes). The optimized conditions in this protocol take important parameters such as temperature, osmolality, and pH into consideration by mimicking the physiological conditions of the fish, thereby enabling physiologically more meaningful results.

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled ...

Neuroscience

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Establishment of an Extracellular Acidic pH Culture System

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In vitro Monitoring of Extracellular pH in Real-Time

Characterizing Mediated Extracellular Electron Transfer in Lactic Acid Bacteria with a Three-Electrode, Two-Chamber Bioelectrochemical System

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Functional Characterization of Na⁺/H⁺ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate