This work was supported by the National Science and Technology Council (MOST 111-2313-B-002-029- and NSTC 112-2313-B-002-050-MY3). For Figure 1, the experimental icons were sourced from BioRender.com.

Two commercial cabbage (B. oleracea var. capitata) cultivars were utilized in this study: &#39;Fuyudori&#39; and &#39;228&#39;. A graphical representation of the protocol workflow is shown in Figure 1. The details of the reagents and the equipment used in this study are listed in the Table of Materials.
1. Preparation of cabbage seedlings
<ol>
	<li>Sow &#39;Fuyudori&#39; and &#39;228&#39; cabbage seeds using commercial plant substrate into round square-shaped holes (D 4.5 cm x H 4.5 cm) of 48-well plug trays (L 49 cm x W 28 cm x H 5 cm). Embed the seeds approximately 1 cm deep into the growing medium.</li>
	<li>Grow cabbage seedlings in a growth room at 22 &#176;C with a 16/8 h light-dark cycle. Maintain the light intensity of 100 &#181;mol m-2&#183;s-1 (white fluorescent tube) during the light phase.</li>
	<li>After 2-3 weeks of growth, use cabbage seedlings at the 2-leaf stage for further mesophyll protoplast isolation. 
	NOTE: The suggested size of cabbage seedlings is a 2-leaf stage, in which the second true leaf is fully expanded, but the third true leaf is not expanded, and its length is shorter than 1 cm (Figure 2A). To synchronize the developmental stages of the cabbage seedlings, &#39;Fuyudori&#39; seeds need to be sown approximately 2 days earlier than &#39;228&#39; seeds due to their different growth rates. This adjustment ensures uniform growth and maturity among the cabbage plants at the desired stage.</li>
</ol>
2. Cabbage protoplasts isolation
<ol>
	<li>Prepare 12.5 mL of enzyme solution (Table 1) before each protoplast isolation.
	<ol>
		<li>Pre-heat a solution with 10 mM MES (pH 5.7) and 0.6 M of mannitol at 55 &#176;C. Continue warming the solution at 55 &#176;C for 10 min after adding 1.5% Cellulase R10 and 0.75% Macerozyme R10.</li>
		<li>After the enzyme solution cools down to room temperature (25 &#176;C), add 10 mM of CaCl2 and 0.1% bovine serum albumin (BSA). Filter sterilize the prepared enzyme solution into a 9 cm Petri dish using a 0.22 &#181;m syringe filter. 
		NOTE: Pre-warming of solution increases enzyme solubility and inactivates protease. The digestion effects vary depending on the different types of cellulase enzymes. The combination of Cellulase R10 and Macerozyme R10 is suitable for cabbage protoplast isolation.</li>
	</ol>
	</li>
	<li>Collect the second newly expanded true leaves from five to eight cabbage seedlings, and slice them into 0.5-1.0 mm strips using a sharp razor blade. Immediately transfer the leaf strips into the freshly prepared enzyme solution. 
	NOTE: Selecting healthy and designated leaves from cabbage at the proper stage is crucial. The second newly expanded true leaves from cabbage seedlings at the 2-leaf stage provide the highest protoplast isolation efficiency. Over-mature plants, cotyledons, or aged leaves are not recommended for protoplast isolation. Five to twelve well-expanded true leaves can successfully yield cabbage protoplasts in 12.5 mL of enzyme solution. The number of leaves required for protoplast isolation depends on the desired quantity of protoplasts needed. Typically, protoplasts obtained from five to eight leaves are sufficient for subsequent experimental procedures.</li>
	<li>After vacuum infiltration in the dark for 30 min (Figure 2B),&#160;keep the cabbage leaf strips immersed in the enzyme solution (Figure 2C) in the dark at room temperature for a further 4-16 h. 
	NOTE: Enzyme digestion time may vary among cabbage cultivars and should be optimized according to the specific genotype. For instance, &#39;Fuyudori&#39; requires a longer digestion time (16 h) compared to &#39;228&#39; (4 h) due to thicker leaves of &#39;Fuyudori&#39;.</li>
	<li>Dilute the protoplasts-containing solution with an equal volume of W5 solution, which consists of 2 mM of MES (pH 5.7), 154 mM of NaCl, 125 mM of CaCl2, 5 mM of KCl, and 5 mM of glucose (see Table 1 for preparation), to stop enzymatic digestion.</li>
	<li>Release the protoplast suspension by gentle swirling or using an orbital shaker. Filter the cell suspension through a 70 &#181;m cell strainer into a 50 mL conical tube. 
	NOTE: The isolated protoplasts are capable of passing through a 70 &#181;m cell strainer, while undigested plant debris is retained by the strainer and subsequently removed from the suspension. Cell strainers can be reused and kept in 75% ethanol after washing, but it is necessary to fully rinse cell strainers with W5 solution to remove residual ethanol before use.</li>
	<li>Centrifuge the protoplasts solution with 150 x g for 2 min at 4 &#176;C then carefully remove the supernatant.&#160;Gently wash the pelleted protoplasts by adding 10 mL of W5 solution along the wall of the conical tube at an approximate flow rate of 1 mL&#183;s-1. Centrifuge the tube at 150 x g for 2 min at 4 &#176;C, and repeat this washing procedure again to ensure complete removal of the enzyme solution.</li>
	<li>Resuspend the protoplasts in the W5 solution and place them on ice for 30 min. After the ice incubation, remove the supernatant by a pipette 1 mL at a time until all the supernatant is removed.</li>
	<li>Resuspend the protoplasts in an ice-pre-chilled MMG solution composed of 4 mM of MES (pH 5.7), 0.4 M of mannitol, and 15 mM of MgCl2 (see Table 1 for preparation).</li>
	<li>Measure the protoplast concentration with a hemocytometer and adjust the final concentration to 4 x 105 protoplasts&#183;mL-1 using MMG solution.</li>
</ol>
3. Protoplasts transfection
<ol>
	<li>Mix a total volume of 10 &#181;L of plasmid (5-10 &#181;g) (Supplementary File 1) with 100 &#181;L of protoplasts (4 x 104 protoplasts) and place on ice for 10 min. 
	NOTE: For transfection-grade plasmid purification, a commercially available plasmid kit is used following the manufacturer&#39;s instructions. Approximately 4 x 104 protoplasts are typically employed in a dual-luciferase reporter assay. For larger-scale experiments, a higher quantity of protoplasts is available for transfection. Specifically, around 2 x 105 protoplasts transfected with 10 &#181;g of plasmid exhibit a proven transfection efficiency ranging from 30% to 40%.</li>
	<li>Add an equal volume (110 &#181;L) of freshly prepared PEG solution (see Table 1 for preparation) containing 40% Polyethylene Glycol 4000 (PEG4000), 0.1 M of CaCl2, and 0.2 M of mannitol into the protoplast solution and mix gently.</li>
	<li>Incubate the protoplast mixture at room temperature in the dark for 10 min, then add 440 &#181;L of W5 solution to terminate the reaction.</li>
	<li>Centrifuge the transfected protoplasts at 150 x g for 2 min at 4 &#176;C and resuspend the pellet in 750 &#181;L of oxygen-enriched W5 solution. Transfer the resuspended protoplasts to a 6-well tissue culture plate pre-coated with 1% BSA.&#160; 
	NOTE: W5 solution used for cell incubation must be&#160;bubbled with oxygen using an oxygen concentrator&#160;(~90% oxygen) connected to an air stone for 5 min (Figure 2D). Following this oxygenation process, the final&#160;dissolved oxygen concentration in the W5 solution increased from 7.84 mg&#183;L-1 &#177; 0.05 mg&#183;L-1 to 29.18&#160;mg&#183;L-1 &#177; 0.43 mg&#183;L-1, as measured with a dissolved&#160;oxygen meter. The volume of W5 solution for resuspension of transfected protoplast depends on the type of tissue cell culture plate used. For 12-well or 24-well cell culture plates, it is advisable to reduce the volume of W5 solution to mitigate hypoxia stress during incubation due to the deeper depth within the wells. For BSA coating, each well of the culture plate received 1 mL of 1% BSA solution, which was allowed to coat the wells for 10 s. The BSA solution was then discarded from each well, and the wells were subsequently refilled with W5 solution. This procedure aimed to create a temporary non-adherent surface using BSA, effectively preventing protoplasts from sticking to the plastic surface of the culture plate during subsequent experimental steps.</li>
</ol>
4. Hypoxia treatment on cabbage protoplasts
<ol>
	<li>Conduct chemical or physically induced hypoxia treatments immediately after protoplast transfection. 
	NOTE: It is recommended that hypoxia treatment be applied immediately after protoplast transfection. Prolonged incubation of protoplasts may diminish the subsequent hypoxia response.
	<ol>
		<li>For chemically induced hypoxia on cabbage protoplasts, add OxyFluor, EC-Oxyrase, and sodium sulfite to the W5 protoplast solution. 
		NOTE: Using 0.6 units&#183;mL-1 EC-oxyrase, 0.6 units&#183;mL-1 OxyFluor, and 1 g&#183;mL-1 sodium sulfite in W5 were the tested conditions capable of inducing BoADH1 and BoSUS1L promoter&#160;(Figure 3A) with low damage to protoplast integrity in the two examined cabbage varieties.</li>
		<li>For oxygen-consuming bag-induced hypoxia, place two oxygen absorber packs in a 3.5 L anaerobic jar to create a hypoxic environment.</li>
	</ol>
	</li>
	<li>Harvest hypoxia-treated protoplasts by centrifuging at 150 x g for 2 min at 4 &#176;C. Freeze the collected protoplasts using liquid nitrogen and store them in a -80 &#176;C freezer for subsequent assays.</li>
</ol>
5. Dual luciferase assay
<ol>
	<li>Perform the dual-luciferase reporter assay according to the manufacturer&#39;s instructions with minor modification.
	<ol>
		<li>Resuspend cabbage protoplasts in 50 &#181;L of 1x passive lysis buffer and vortex for 10 s.</li>
		<li>Incubate disrupted protoplasts on ice for 10 min and collect the supernatant after centrifugation at 10,000 x g for 10 min at 4 &#176;C.</li>
		<li>Add 20 &#181;L of cell lysate to each well of the microplate. Dispense 100 &#181;L of Luciferase assay reagent II into the wells and measure firefly luciferase activity using a microplate reader.</li>
		<li>Add 100 &#181;L of the commercially available assay reagent to each well and measure Renilla luciferase activity using a microplate reader.</li>
	</ol>
	</li>
</ol>

Efficient Cabbage Protoplast System for Studying Flooding Tolerance Mechanisms

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The authors declare no competing interest.

This protocol presents a streamlined method for protoplast isolation from two commercial cabbage cultivars. The efficacy of this method is primarily assessed through two critical quality control parameters: the yield of viable protoplasts and the efficiency of protoplast transfection. Implementing this protocol resulted in a yield exceeding 4.00 x 106 protoplasts&#183;g&#8722;1&#183;FW of mesophyll tissue from both cabbage cultivars (Figure 2E,F). This ...

Global climate change has exacerbated flooding, which has emerged as an increasingly critical issue worldwide. Recent decades have witnessed an upward trend in the frequency of flooding events, resulting in substantial crop losses1,2. Cabbage (Brassica oleracea var. capitata L.), a vegetable of significant global importance, is susceptible to the adverse effects of heavy rainfall, necessitating the development of flooding-tolerant cabbage cultivars to ensure sustainable production in the face of extreme weather events. Therefore, understanding the molecular mechanisms associated with flooding stress in cabbage is essential to meet this challenge.
To understand gene regulation mechanisms in plants under submerged conditions, transgenic lines are widely used for genetic functional studies. However, this approach is constrained by high costs, time-intensive transformation, and subculture processes, as well as low transformation efficiencies in many crop species, necessitating the development of alternative methodologies. Protoplast-based transient expression systems have been widely applied in plant molecular research as a versatile and efficient alternative. These systems facilitate investigations into promoter activity, signaling pathways in response to environmental cues, protein-protein or protein-DNA interactions, and subcellular localization3. The establishment of protoplast transient expression systems has been reported not only in model plants4,5 but also in economically important crops such as sugarcane6, carnation7, Phalaenopsis orchids8, and eggplant9. Moreover, these systems have been successfully implemented in woody plants, including Camellia oleifera10and Populus trichocarpa11. However, protocols for applying protoplast systems to study leafy vegetables under submergence-induced hypoxia stress are limited. Therefore, an integrated protocol has been developed in this work for those interested in studying hypoxia responses in leafy vegetables using a cabbage protoplast transient expression system.
To carry out the submergence-induced hypoxia response at the cellular level, several oxygen-scavenging methodologies have been employed in previous studies to simulate hypoxic environments. These include the use of EC-Oxyrase, OxyFluor, sodium sulfite, and oxygen-consuming bags. EC-Oxyrase is typically used for anaerobic treatment in human cell lines12 and Arabidopsis protoplasts13. OxyFluor has been found to be effective in mitigating photobleaching caused by reactive oxygen species during live-cell fluorescence imaging14,15. Sodium sulfite has been employed in the anaerobic treatment of nematodes16 and, more recently, in rice protoplasts, in conjunction with techniques such as chromatin immunoprecipitation (ChIP) assays17. Oxygen absorber packs, primarily used for anaerobic bacterial culture18, have also demonstrated efficacy in inducing the activation of ZmPORB1 promoters in maize protoplasts under anaerobic conditions19.
The present work aims to establish a robust pipeline for cabbage protoplast isolation and transient expression. Subsequently, the efficacy of various oxygen-adjusting treatments was evaluated by assessing the promoter activity of anaerobic response genes using dual-luciferase assays. The protocol developed in this study is anticipated to be valuable for future research related to submergence or hypoxia stress in Brassica systems.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-(N-morpholino) ethanesulfonic Acid (MES)</td>
			<td>PhytoTech Labs</td>
			<td>M825</td>
			<td>For enzyme solution preparation</td>
		</tr>
		<tr>
			<td>228 cabbage seeds</td>
			<td>Takii &#38; Co., Ltd. (Kyoto, Japan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Tube</td>
			<td>SPL Life Sciences</td>
			<td>50050</td>
			<td>For enzyme solution preparation</td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Alpha Plus</td>
			<td>16106</td>
			<td>For protoplast incubation</td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>Sorfa&#160;</td>
			<td>SCS701</td>
			<td>For protoplast filtration</td>
		</tr>
		<tr>
			<td>9-cm Petri dish</td>
			<td>Alpha Plus</td>
			<td>16001</td>
			<td>For enzymatic digestion</td>
		</tr>
		<tr>
			<td>Anaerobic jar</td>
			<td>HIMEDIA</td>
			<td>Anaerobic Jar 3.5 L</td>
			<td>For hypoxia treatment&#160;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td>For W5 solution preparation and culture plate coating</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>J.T.Baker</td>
			<td>131301</td>
			<td>For W5 solution and PEG solution preparation</td>
		</tr>
		<tr>
			<td>Cellulase R10</td>
			<td>Yakult</td>
			<td></td>
			<td>For enzyme solution preparation</td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Tarsons&#160;</td>
			<td>402030</td>
			<td>For vacuum infiltration</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Bioshop</td>
			<td>GLU501</td>
			<td>For W5 solution preparation</td>
		</tr>
		<tr>
			<td>Dissolved oxygen meter</td>
			<td>Thermo Scientific</td>
			<td>Orion Star A223</td>
			<td>For oxygen measurement</td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M1902</td>
			<td>For enzyme solution, PEG solution, and MMG solution preparation</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1960</td>
			<td>For Dual-luciferase reporter assay</td>
		</tr>
		<tr>
			<td>EC-Oxyrase</td>
			<td>Oxyrase Inc.</td>
			<td>EC-0005</td>
			<td>For hypoxia treatment</td>
		</tr>
		<tr>
			<td>Fuyudori cabbage seeds</td>
			<td>Kobayashi Seed Co., Ltd. (Kakogawashi, Japan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-Speed refrigerated centrifuge</td>
			<td>Hitachi</td>
			<td>CR21GIII</td>
			<td>For protoplast harvest</td>
		</tr>
		<tr>
			<td>Macerozyme R10&#160;</td>
			<td>Yakult</td>
			<td></td>
			<td>For enzyme solution preparation</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Alfa Aesar</td>
			<td>12315</td>
			<td>For MMG solution preparation</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Hitachi</td>
			<td>CT15RE</td>
			<td>For protoplast harvest</td>
		</tr>
		<tr>
			<td>Microplate</td>
			<td>Greiner</td>
			<td>655075</td>
			<td>For Dual-luciferase reporter assay</td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax Mini</td>
			<td>For Dual-luciferase reporter assay</td>
		</tr>
		<tr>
			<td>Millex 0.22 &#956;m syringe filter</td>
			<td>Merck</td>
			<td>SLGP033RS</td>
			<td>For enzyme solution preparation</td>
		</tr>
		<tr>
			<td>Oil Free Vacuum Pump</td>
			<td>Rocker&#160;</td>
			<td>Rocker 300</td>
			<td>For vacuum infiltration</td>
		</tr>
		<tr>
			<td>OxyFluor</td>
			<td>Oxyrase Inc.</td>
			<td>OF-0005</td>
			<td>For hypoxia treatment</td>
		</tr>
		<tr>
			<td>Oxygen absorber pack</td>
			<td>Mitsubishi Gas Chemical Company</td>
			<td>AnaeroPack, MGCC1</td>
			<td>For hypoxia treatment</td>
		</tr>
		<tr>
			<td>Oxygen concentrator</td>
			<td>UTMOST PERFECT</td>
			<td>AII-X</td>
			<td>For oxygen-bubbling in W5 solution</td>
		</tr>
		<tr>
			<td>Plant substrate</td>
			<td>Klasmann-Deilmann</td>
			<td>Potgrond H substrate</td>
			<td>For cabbage seedlings preparation</td>
		</tr>
		<tr>
			<td>Plasmid Midi Kit</td>
			<td>QIAGEN</td>
			<td>12145</td>
			<td>For purification of transfection-grade plasmid DNA&#160;</td>
		</tr>
		<tr>
			<td>Polyethylene Glycol 4000</td>
			<td>Fluka</td>
			<td>81240</td>
			<td>For protoplast transfection</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>J.T.Baker</td>
			<td>304001</td>
			<td>For W5 solution preparation</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Gillette</td>
			<td></td>
			<td>For cabbage leaf strips preparation</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Bioshop</td>
			<td>SOD002</td>
			<td>For W5 solution preparation</td>
		</tr>
		<tr>
			<td>Sodium sulfite</td>
			<td>Sigma-Aldrich</td>
			<td>S0505</td>
			<td>For hypoxia treatment</td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Yihder</td>
			<td>BU-240D</td>
			<td>For enzyme solution preparation</td>
		</tr>
	</tbody>
</table>

development of a cabbage protoplast system for studying hypoxia tolerance in brassica

As climate change brings more heavy rainfall, cabbage, a key Brassicaceae vegetable, faces significant yield losses due to flooding-induced hypoxia stress. To identify mechanisms of flooding tolerance in cabbages, a versatile platform for genetic functional studies is needed to overcome the transformation-recalcitrant nature of cabbages. In this study, a cabbage protoplast transient expression system and a corresponding protoplast hypoxia induction protocol were developed. This protocol achieved a high yield and integrity of protoplast isolation from cabbage leaves, with a transfection efficiency exceeding 40% using optimized enzymatic conditions. To alleviate potential hypoxic influence before treatments, the W5 solution was bubbled with oxygen gas to increase dissolved oxygen levels. Several chemicals for adjusting oxygen levels and physiological oxygen-scavenging treatments were tested, including EC-Oxyrase, OxyFluor, sodium sulfite, and an oxygen absorber pack. Dual-luciferase assays showed that promoters of anaerobic respiration response genes BoADH1 and BoSUS1L were activated in cabbage protoplasts after hypoxia treatments, with the highest induction level observed after treatment with the oxygen absorber pack. In summary, the cabbage protoplast transient expression system combined with hypoxia treatment demonstrates an efficient and convenient platform. This platform can facilitate studies of gene function and molecular mechanisms associated with hypoxia responses in cabbages.

This work successfully developed a transient expression system utilizing cabbage protoplasts (see Figure 1 for workflow). Protoplasts were isolated from 2- to 3-week-old cabbage true leaves of appropriate size (Figure 2A) from commercial cabbage cultivars &#39;Fuyudori&#39; and &#39;228&#39; using cellulase/macerozyme digestion and dark vacuum infiltration (Figure 2B,C). To mitigate the hypoxic conditions experience...

Development of a Cabbage Protoplast System for Studying Hypoxia Tolerance in Brassica

This protocol describes an efficient cabbage mesophyll protoplast system. Various oxygen-deficient treatments were tested, and the system showed a high activation of hypoxia-responsive genes, facilitating studies of the genetic and molecular mechanisms of flooding tolerance in Brassicaceae vegetables.

As climate change brings more heavy rainfall, cabbage, a key Brassicaceae vegetable, faces significant yield losses due to flooding-induced hypoxia ...

development-cabbage-protoplast-system-for-studying-hypoxia-tolerance

Research

JoVE Journal

Biology

316 Views.  National Taiwan University. This protocol describes an efficient cabbage mesophyll protoplast system. Various oxygen-deficient treatments were tested, and the system showed a high activation of hypoxia-responsive genes, facilitating studies of the genetic and molecular mechanisms of flooding tolerance in Brassicaceae vegetables.

Video: Development of a Cabbage Protoplast System for Studying Hypoxia Tolerance in Brassica

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...