This work was supported by the National Natural Science Foundation of China (32071174, 32200941, 32371183, and 31771312).

All methods described here have been approved by the animal ethical committee at Beijing Sport University and carried out in compliance with the National Institutes of Health (NIH) guidelines and the Chinese animal protection laws and institutional guidelines.
1. Mating and feeding rats
<ol>
	<li>Purchase specific pathogen-free (SPF) rated 10-week-old Wistar female rats and 11-week-old Wistar male rats.</li>
	<li>Implement adaptive feeding: Feed the Wistar rats ad libitum with a standard rodent diet and provide access to tap water for one week in the designated feeding facility. Ensure all rats are housed in a 12-h light-dark cycle at a constant temperature of 22 &#176;C and humidity of 45-55%.</li>
	<li>Examine the estrous of the female rat.
	<ol>
		<li>Prepare the equipment and materials, including clean glass microscope slides, 2 mm diameter cotton swabs, saline solution (0.9% NaCl), and microscope.</li>
		<li>Label each microscope slide with the date and rat identification.</li>
		<li>Between 6 and 7 PM, to minimize stress, restrain the female rat gently. Grasp the base of the tail with the thumb and forefinger while applying pressure from the other fingers onto the lumbar vertebra of the rat.</li>
		<li>Lift the tip of the tail and expose the vaginal. Moisten a cotton swab with saline solution, then put the tip into the vagina and gently roll the tip to collect vaginal secretions.</li>
		<li>Roll the swab tip across the slide, avoiding reapplication to the same area.</li>
		<li>Examine the smear under the microscope and use the low magnification objective (10x) to locate and observe the cells. 
		NOTE: During estrus, the vaginal secretions have larger epithelial nuclei vanished, replaced by squamous exfoliated keratinized epithelial cells that accumulated into a mound.</li>
		<li>Record the stage of the estrous cycle for each rat.</li>
	</ol>
	</li>
	<li>Between 7 and 8 PM, place a male and a female rat in the mating cage. 
	NOTE: The female rats need to be in proestrus or estrus for mating.</li>
	<li>On the second day, between 7 and 8 AM, inspect the mating cage for copulatory plugs. If a copulatory plug is found at the base of the mating cage, proceed to create a vaginal secretion smear for the female rat following the same method as described in steps 1.3.4- 1.3.6. 
	NOTE: Copulatory plugs are milky white or yellowish gelatinous materials made of male rat seminal vesicle and prostate secretions combined with vaginal secretions.</li>
	<li>Confirm pregnancy in the female rat:&#160;after finding copulatory plugs in the mating cage, observe sperm on the vaginal secretion smear. Designate this day as gestation day 1 (GD1).</li>
	<li>Isolate the pregnant rat in a separate cage. 
	NOTE: During the initial stages of pregnancy, it is imperative to prevent a pregnant rat from encountering unfamiliar male chemical cues in its environment or from cohabiting with an unfamiliar male rat.</li>
	<li>Rat gestation periods typically last 21-23 days. Measure and record the rats&#39; body weights from GD1 to GD20.</li>
	<li>On GD20, replace the pregnant rat&#39;s cage with a clean one and place medical absorbent cotton (similar in size to the rat) in the cage. Supply sufficient water and food for seven days&#39; survival, and position the cage in a quiet location. 
	NOTE: Do not change the cage for at least 1 week after rat parturition. When switching to a clean cage, transfer the used absorbent cotton from the dirty cage into the new one to ensure the rats can recognize the scent of their offspring.</li>
	<li>On the 21st day postpartum, separate the offspring by gender and place them into two cages. 
	NOTE: Due to the smaller size of the 21-day-old offspring, place the hard feed pellets in a glass petri dish inside the cage.</li>
</ol>
2. Prenatal swimming training program
<ol>
	<li>Conduct adaptation training for all experimental female rats for 5 days.
	<ol>
		<li>Prepare a transparent plastic container at least 20 cm in height and fill it with 10 cm of water at 34 &#177; 1&#176;C.</li>
		<li>Grasp the tip of the rat&#39;s tail and lift it, then place the rat in the water.</li>
		<li>During the 15-min adaptive training, closely monitor the water temperature every 5 min, adding warm water to maintain the temperature.</li>
		<li>After the training, transfer the rat to clean water at 34 &#177; 1&#176;C to wash its fur. Then, transfer the rat to a cage with towels and use a hairdryer to dry its fur. 
		NOTE: Noise and a hot environment can cause stress to rats. Therefore, it is recommended to use a temperature-adjustable and quiet hair dryer.</li>
		<li>Kindly return the rat to its respective cages.</li>
	</ol>
	</li>
	<li>Rat mating (follow the steps 1.3-1.6).</li>
	<li>After step 1.7, randomly assign pregnant rats into a sedentary group (SED) and an exercise group (EX).</li>
	<li>Weigh the rat daily before every training session. 
	NOTE: During the adaptive training, the weight loss of rats should not exceed 3 g/day. During the prenatal training, the weight of the rats should gradually increase. If the rats lose more than 3 g per day, reduce the daily training time by 50% to allow for recovery.</li>
	<li>Conduct prenatal swimming training for the EX from GD1 to GD20, 6 days per week, from 8:00 to 9:00 in the morning.
	<ol>
		<li>Prepare a circular water bucket with a diameter of 50 cm and a minimum height of 50 cm and fill it with 40 cm of water at a temperature of 34 &#177; 1&#176;C.</li>
		<li>From GD1 to GD5, initially perform the swimming training for 20 min a day and progressively increase to 60 min a day.</li>
		<li>From GD6 to GD20, continue the exercise training for 60 min per day for 6 days per week.</li>
		<li>For aftercare, refer to steps 2.1.4-2.1.5.</li>
		<li>After exercise, give the EX rat 5 g of rodent sunflower seeds to provide nutritional support.</li>
	</ol>
	</li>
	<li>During the EX training every day, place the SED rat in the same water environment as adaptive training (step 2.1.1). Let the SED rat spend the same time in the water environment as the EX rat. After exercise, give the SED rat 5 g of rodent sunflower seeds to provide them with nutritional support and soothing emotions.</li>
	<li>After each swim training session, clean and disinfect all equipment with ultraviolet light, including water buckets and towels.</li>
</ol>
3. Physical characteristics of fetuses
<ol>
	<li>On the morning of GD20.5, anesthetize the female rat with 3% isoflurane and euthanize it in a CO2 chamber.</li>
	<li>Use a surgical knife to make an incision in the lower abdomen of the rat. Make sure that the incision slants towards both sides of the abdomen, ensuring complete exposure of the uterus.</li>
	<li>Carefully open the uterus using forceps and scissors to expose the fetuses and placentas. Gently separate the thin amnion surrounding the fetuses using forceps to avoid scissor-induced damage to the fetuses and placentas.</li>
	<li>Clean the bodies of fetuses using sterile gauze to remove blood and mucus. Sequentially separate fetuses and placentas by their anatomical positions, then weigh them.</li>
	<li>Observe the distance between the anus and genitalia to determine the gender of the fetuses. Male fetuses have a greater distance than females. Record the gender and quantity of fetuses for future reference.</li>
	<li>Measure the length of the fetuses using a caliper while lying flat. Because the fetuses are still alive, they may curl up after being dissected, so unfold their bodies to take measurements. 
	NOTE: To ensure the consistency of experimental results and the measurement methods are consistent, the same experimenter maintains the same standards throughout. Fetuses were euthanized by institutional recommended guidelines and tissue samples were collected for further analysis as needed.</li>
</ol>

Exploring Maternal Exercise for the Well-Being of Rat Offspring

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This study aims to establish a feasible exercise program for pregnant rats, including detailed rat mating methods, swimming training protocols, and research methods to evaluate physiological indicators in fetuses. We have developed a prenatal swimming exercise training model through practice, with corresponding solutions to potential issues that may arise in the protocol (Table 1). This protocol will make it more practical and assist researchers in establishing prenatal exercise models.
<p class="jov...

The Developmental Origins of Health and Disease (DOHaD) concept emerged over the last two decades, which has shown that environmental influences during early development affect the risk of later pathophysiological processes associated with chronic non-communicable disease (NCD), especially diabetes, cardiovascular disease, and some types of cancer1. During the most plastic phase of fetal development, where offspring are exposed to the intrauterine environment, the gene and environment interaction and uteroplacental perfusion are crucial factors of fetal reprogramming2. Previous studies have predominantly utilized animal models to investigate the detrimental effects, such as maternal undernutrition, overnutrition, or obesity during pregnancy, and prenatal exposure to chemicals or hypoxia, on fetal development and long-term phenotypic effects in the offspring3. This produced low birth weight and later growth and produced adverse effects on offspring, including elevated blood pressure, dyslipidemia, and insulin resistance4. Since the rate of overweight and obesity is rapidly increasing among women of reproductive age, establishing effective interventions to prevent the intergenerational transmission of these deleterious maternal disorders is urgently needed, which has an important impact on population health5,6.
Exercise has long been recognized as an important preventive therapeutic for type 2 diabetes, hypertension, and several other diseases7. Exercise during pregnancy has beneficial effects for the mother and confers beneficial effects on offspring, thus reducing the maternal transmission of disease to offspring8. Several years of research have demonstrated exercise&#39;s safety and profound benefits, including substantial reductions in common pregnancy conditions such as gestational diabetes mellitus9,10. Regarding maternal and fetal safety, conducting exercise training and post-nursing protocols is challenging.
David11 et al. described animal exercise models applicable to cardiovascular health research. The three commonly used exercise models, voluntary wheel running, motorized treadmills, and swimming, have advantages and disadvantages. The voluntary wheel running closely mimics the locomotor behavior of rats, enabling them to move according to their circadian rhythm. However, this sort of exercise needs more precise regulation of intensity and duration. The motorized treadmill can control exercise intensity, volume, and duration, but sometimes it needs electric stimulation, which may trigger physical and psychological stress in the animals. Swimming is widely used in rodent studies due to the inherent swimming ability of rats, which can also avoid electric stimulation or mechanical damage to their feet and tails12. In studies on prenatal exercise models13,14, swimming is a commonly used exercise modality. As pregnancy progresses and the abdomen of the rat swells, treadmill and wheel running exercises may cause repeated contact of the abdomen with hard surfaces. In contrast, the water environment in swimming provides buoyancy, reducing the risk of exercise-related injuries for pregnant animals.
This protocol presents the rat breeding methods, how to perform the swimming training during pregnancy, and the post-breeding nursing protocols for pregnant rats after training. This pregnant swimming training model can be applied to a wide range of rat and mouse species, which may thus provide a useful rodent model for future investigation using animal-based models to study the mechanisms regulating the beneficial effects of maternal exercise on offspring health and the benefits of exercise across generations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Caliper</td>
			<td>Mitutoyo Measuring Instruments (Shanghai) Co., Ltd</td>
			<td>530-101 N15</td>
			<td>Fetus dissect</td>
		</tr>
		<tr>
			<td>Circular water bucket</td>
			<td>Naliya</td>
			<td>N/A</td>
			<td>Rat swimming</td>
		</tr>
		<tr>
			<td>Experimental Surgical Instrument&#160;</td>
			<td>Shenzhen RWD Life Technology Co., LTD</td>
			<td>SP0001-G</td>
			<td>Fetus dissect</td>
		</tr>
		<tr>
			<td>Glass Microscope Slides</td>
			<td>Jiangsu Shitai experimental equipment Co., LTD</td>
			<td>80312-3161</td>
			<td>Vaginal smear</td>
		</tr>
		<tr>
			<td>Glass petri dish</td>
			<td>Merck Life Technologies</td>
			<td>BR455751-10EA</td>
			<td>Offspring rearing</td>
		</tr>
		<tr>
			<td>Hairdryer</td>
			<td>Panasonic</td>
			<td>EH-WNE5H</td>
			<td>Post-swimming care</td>
		</tr>
		<tr>
			<td>Information Card</td>
			<td>Zhongke Life Science</td>
			<td>SS3</td>
			<td>Rat mating</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Shenzhen RWD Life Technology Co., LTD</td>
			<td>R510-22-10</td>
			<td>Fetus dissect</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus</td>
			<td>IX71-F22PH</td>
			<td>Vaginal smear</td>
		</tr>
		<tr>
			<td>Mating cage</td>
			<td>Zhongke Life Science</td>
			<td>SS3</td>
			<td>Rat mating</td>
		</tr>
		<tr>
			<td>Medical Absorbent Cotton</td>
			<td>Hongxiang Sanitary Materials Co., LTD</td>
			<td>N/A</td>
			<td>The pregnant rats are anticipating giving birth</td>
		</tr>
		<tr>
			<td>Rodent Anesthesia Machine, Gas Anesthesia</td>
			<td>Shenzhen RWD Life Technology Co., LTD</td>
			<td>R500IE</td>
			<td>Fetus dissect</td>
		</tr>
		<tr>
			<td>Rodent breeding feed</td>
			<td>Beijing Huafukang Biotechnology Co., LTD</td>
			<td>1032</td>
			<td>Pregnant rat feeding</td>
		</tr>
		<tr>
			<td>Rodent maintenance feed</td>
			<td>Beijing Huafukang Biotechnology Co., LTD</td>
			<td>1022</td>
			<td>Offspring rearing</td>
		</tr>
		<tr>
			<td>Rodent sunflower seeds</td>
			<td>Jolly</td>
			<td>JP241</td>
			<td>Nutritional supplement</td>
		</tr>
		<tr>
			<td>Soundproof cotton</td>
			<td>Kufu Medical Instrument</td>
			<td>N/A</td>
			<td>The pregnant rats are anticipating giving birth</td>
		</tr>
		<tr>
			<td>Sterile Cotton Swab (2 mm diameter)</td>
			<td>Kufu Medical Instrument</td>
			<td>N/A</td>
			<td>Vaginal smear</td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Kufu Medical Instrument</td>
			<td>N/A</td>
			<td>Fetus dissect</td>
		</tr>
		<tr>
			<td>Stroke-physiological Saline Solution (0.9% NaCl)</td>
			<td>Shandong Hualu Pharmaceutical Co., LTD</td>
			<td>N/A</td>
			<td>Vaginal smear</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Beekman organism</td>
			<td>N/A</td>
			<td>The monitoring of rat swimming</td>
		</tr>
		<tr>
			<td>Towels</td>
			<td>Grace</td>
			<td>N/A</td>
			<td>Post-swimming care</td>
		</tr>
		<tr>
			<td>Transparent plastic container</td>
			<td>Naliya</td>
			<td>N/A</td>
			<td>Swimming adaptive training</td>
		</tr>
		<tr>
			<td>Ultraviolet light</td>
			<td>Merck Life Technologies</td>
			<td>Z169633-1EA</td>
			<td>Post-swimming care</td>
		</tr>
		<tr>
			<td>Water heater</td>
			<td>Haier</td>
			<td>EC6001-Q6S</td>
			<td>Rat swimming</td>
		</tr>
		<tr>
			<td>Weight Scale</td>
			<td>Electronlc Acale</td>
			<td>JM.A10001</td>
			<td>Body weight measurement</td>
		</tr>
		<tr>
			<td>Wistar Rats</td>
			<td>Vital river Laboratory Animal Technology Co., LTD</td>
			<td>N/A</td>
			<td>Experiment</td>
		</tr>
	</tbody>
</table>

swimming exercise protocol and care methods for pregnant rats

The developmental origins of health and disease concept highlights the impact of early environments on chronic non-communicable diseases like diabetes, cardiovascular disease, and cancer. Studies using animal models have investigated how maternal factors such as undernutrition, overnutrition, obesity, and exposure to chemicals or hypoxia affect fetal development and offspring health, leading to issues like low birth weight, high blood pressure, dyslipidemia, and insulin resistance. Given the increasing prevalence of overweight and obesity among reproductive-age women, effective interventions are critical. Maternal exercise during pregnancy has emerged as a key intervention, benefiting both mother and offspring and reducing the risk of disease. This study compares the differences of three exercise models on pregnant rats: voluntary wheel running, motorized treadmills, and swimming. Swimming is the most beneficial option due to its safe and controlled intensity levels. This protocol details the rat breeding methods, swimming training during pregnancy, and post-breeding nursing protocols. This model, suitable for various rat and mouse species, is useful for studying the benefits of maternal exercise on offspring health and intergenerational wellness.

In the rat breeding methods section (Figure 1), we determine the estrous cycle of female rats by making vaginal secretion slides to increase the success rate of breeding. The rats&#39; estrous cycle lasts 4-5 days, including proestrus, estrus, metestrus, and diestrus. Female rats in metestrus or diestrus exhibit behaviors such as distancing themselves from males. At the same time, those in estrus are willing to mate, and ovulation in female rats mostly occurs at the end of estrus. During pro...

Read this Scientific Article Protocol about Author Spotlight: Enhancing Offspring Health with Maternal Exercise During Pregnancy at JoVE.com

Author Spotlight: Enhancing the Offspring Health in Rats with Maternal Exercise During Pregnancy

Here, we present a protocol to delineate rat breeding methods, swimming training procedures, and post-breeding nursing protocols for pregnant rats after training. This protocol provides an animal model for studying the effects of maternal exercise during pregnancy on offspring and its underlying mechanisms.

Swimming Exercise Protocol and Care Methods for Pregnant Rats

The developmental origins of health and disease concept highlights the impact of early environments on chronic non-communicable diseases like diabetes, ...

swimming-exercise-protocol-and-care-methods-for-pregnant-rats

Research

JoVE Journal

Developmental Biology

607 Views.  Beijing Sport University. Here, we present a protocol to delineate rat breeding methods, swimming training procedures, and post-breeding nursing protocols for pregnant rats after training. This protocol provides an animal model for studying the effects of maternal exercise during pregnancy on offspring and its underlying mechanisms.

Video: Author Spotlight: Enhancing the Offspring Health in Rats with Maternal Exercise During Pregnancy

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about the effect of natural uranium on the behavior of bone cells. In particular, the impact of uranium on osteoclasts, the cells responsible for the resorption of the bone matrix, is not documented. To investigate this issue, we have established a new protocol using uranyl acetate as a source of natural uranium and the murine RAW 264.7 cell line as a model of osteoclast precursors. Herein, we detailed all the assays required to test uranium cytotoxicity on osteoclast precursors and to evaluate its impact on the osteoclastogenesis and on the resorbing function of mature osteoclasts. The conditions we have developed, in particular for the preparation of uranyl-containing culture media and for the seeding of RAW 264.7 cells allow to obtain reliable and highly reproductive results. Moreover, we have optimized the use of software tools to facilitate the analysis of various parameters such as the size of osteoclasts or the percentage of resorbed matrix.

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Uranium is known to affect bone metabolism. Here, we present a protocol aimed at investigating the effect of natural uranium exposure on the viability, the differentiation, and the function of osteoclasts, the cells in charge of bone resorption.

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about ...

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended effects that extrinsic patterning factors might introduce into human pluripotent stem cell (hPSC) models of brain development or pathophysiology, such as masking disease phenotype. Here, we present two cerebellar differentiation protocols for hPSCs, designed with simpler startup method, fewer patterning factors, and less material requirements than previous protocols. Recently, we developed culture procedures, which generate free-floating 3-dimensional (3D) products consistent with other brain &#34;organoid&#34; protocols, including morphologies relevant to modeling brain development such as sub/ventricular zone- and rhombic lip-like structures. The second uses an adherent, 2D monolayer procedure to complete differentiation, which is shown capable of generating functional cerebellar neurons, as products are positive for cerebellar-associated markers, and exhibit neuron-like calcium influxes. Together, these protocols offer scientists a choice of options suited to different research purposes, as well as a basic model for testing other types of streamlined neural differentiations.

We describe a simplified 3D differentiation protocol for hPSCs, using defined medium and reduced growth factors, capable of generating cell aggregates with early neuroepithelial structures and positive for cerebellar-associated markers, as well as an optional 2D modification for differentiating cells as a monolayer to generate functional neurons.

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...