This work was supported by the National Natural Science Foundation of China (81973569, 82274207 and 82104533), the Xinglin Scholar Research Promotion Project of Chengdu University of TCM (XKTD2022013), and the Key Research and Development Program of Ningxia (2023BEG02012).

Rhodiola crenulata is collected from Zhuoda Snow Mountain, Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province, China (N 31.44570&#176;, E 99.96086&#176;, 4892 m). The plants are authenticated as genuine by Professor Yi Zhang at the School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine.
1. Collection of Rhodiola crenulata
<ol>
	<li>Photograph the habitat map of Rhodiola crenulata.</li>
	<li>Photograph the whole plant, leaves, calyx, and rhizome of Rhodiola crenulata.</li>
	<li>Use a spade to clear the weeds and broken stones within 1 m of the Rhodiola crenulata to ensure the subsequent smooth mining.</li>
	<li>Dig up the soil with a hoe until the whole rhizosphere is seen and collect the taproot. 
	NOTE:The roots and rhizomes of Rhodiola crenulata used in medicinal parts should be collected in autumn, when the flower stems wither.</li>
</ol>
2. Characteristic identification
<ol>
	<li>Observe the appearance traits of Rhodiola crenulata with the unaided eye: cylindrical and short taproots and rhizomes, brown surface, membranous yellow epidermis with a pink pattern, and orange-red or burgundy slices.</li>
	<li>Identify it by smell: It gives a fragrant smell when near the nose.</li>
	<li>Identify it by taste: Take a small piece of root into the mouth, first sip and then chew, taste slightly bitter, then sweet.</li>
</ol>
3. Microscopic identification of starch granules in medicinal powder
<ol>
	<li>Remove the soil on the surface of the Rhodiola crenulata with a brush, put it in the oven at 40 &#176;C, and turn the herbs every 24 h. 
	NOTE: The ease of breaking of medicinal materials is considered the standard for drying moisture.</li>
	<li>Powder the dried medicinal materials using a powder machine and filter the medicinal powder using the medicated No. 3 sieve (see Table of Materials).</li>
	<li>Take a clean slide (see Table of Materials), dig the powder with a dissecting needle (see Table of Materials), and place it evenly at one-third of the slide within 2 mm.</li>
	<li>Use a glass dropper (see Table of Materials) to add a drop of deionized water to the powder. Use a tweezer (see Table of Materials) to hold one end of the cover glass (see Table of Materials) to touch the liquid level quickly and cover the powder. 
	NOTE: Use an anatomical needle to gently mix water and medicinal powder to ensure uniform sample mixing. There should be no air bubbles between the slide, powder, and cover glass.</li>
	<li>Open the microscope (see Table of Materials) and place the slide in step 3.4 onto the platform to secure it. Adjust the light source and the coarse focus spiral to see the powder. Adjust the fine parafocal spiral until the tissues are seen clearly. Switch to a 40x objective and observe the starch granules. 
	&#8203;NOTE: Starch the grains presenting as single or multiple grains, and the umbilical point appears herringbone or crack-shaped.</li>
</ol>
4. Microscopic identification of catheters, cork cells, fibers, wood parenchyma cells, and pigment masses in medicinal powder
<ol>
	<li>Take a clean slide (see Table of Materials), dig the powder with a dissecting needle (see Table of Materials), and place it at one-third of the slide.</li>
	<li>Use a glass dropper (see Table of Materials) to add a drop of chloral hydrate (see Table of Materials) to the powder. Take the slide with a tweezer (see Table of Materials) and heat it in the alcohol lamp three times, each time for 1 s. 
	NOTE: Bubbles should be avoided during heating. The liquid remains non-flowing, indicating that the penetration is complete.</li>
	<li>Use a glass dropper to add a drop of glycerin (see Table of Materials). Use a tweezer to hold one end of the cover glass to touch the liquid level quickly.</li>
	<li>Open the microscope and place the slide onto the platform to secure it. Adjust the light source and the coarse focus spiral to see the powder. Adjust the fine parafocal spiral until the tissues are seen clearly. Observe the catheter, cork cells, fibers, wood parenchyma cells, and pigment block by switching to a 40x objective lens. 
	&#8203;NOTE: Polygonal or long polygonal of cork cells, spiral vessel with obvious helical structure, xylem parenchyma containing calcium oxalate sand crystals, and red or brownish-red pigment block.</li>
</ol>
5. Preparation of thin layer chromatographic (TLC) samples of Rhodiola crenulata and its reference
<ol>
	<li>Place the weighing paper on the balance (see Table of Materials) and weigh 3 g powder of Rhodiola crenulata.</li>
	<li>Take the powder into a 100 mL conical bottle (see Table of Materials), and add 25 mL of methanol with a big belly pipette (see Table of Materials). Put the conical bottle into the ultrasonic instrument. Set the power to 250 W, frequency to 40 kHz, and time to 30 min (see Table of Materials), and switch on the instrument. 
	NOTE: The purpose of the ultrasound instrument is to ensure that the powder of Rhodiola crenulata is completely dissolved without affecting the results of subsequent thin-layer chromatographic experiments.</li>
	<li>Remove the conical bottle and rinse the outer bottle with running water to room temperature (RT).</li>
	<li>Aspirate 800 &#181;L of liquid prepared in step 5.3 with a 1 mL syringe. Filter with 0.22 &#181;m microporous filter membrane (see Table of Materials) and collect 400 &#181;L of midstream sample solution of Rhodiola crenulata in a chromatographic sample bottle.</li>
	<li>Weigh and add 2 mg of salidroside, tyrosol, and gallic acid (see Table of Materials) into 3 separate 100 mL conical bottles, respectively. Add 25 mL of methanol with a big belly pipette into every conical bottle.</li>
	<li>Put the conical bottle into the ultrasonic instrument, set the power to 250 W, frequency to 40 kHz, and time to 30 min, and repeat step 5.3 (see Table of Materials).</li>
	<li>Aspirate 800 &#181;L of liquid prepared in step 5.6 with a 1 mL syringe. Filter with 0.22 &#181;m microporous filter membrane (see Table of Materials) and collect 400 &#181;L of midstream salidroside, tyrosol, and gallic acid standard solution in corresponding chromatographic sample bottles.</li>
</ol>
6. TLC identification
<ol>
	<li>Pipette trichloromethane (5 mL), ethyl acetate (4 mL), methanol (2 mL), and formic acid (0.5 mL) (see Table of Materials). Add to one side of the double-tank chromatography cylinder (see Table of Materials), shake, and mix evenly. Cover the upper cylinder head.</li>
	<li>Place the gallic acid, salidroside, tyrosol standard solution, and Rhodiola crenulata solution on the sample rack in positions A1-A4.</li>
	<li>Place the 5 cm x 10 cm silicone sheet (see Table of Materials) on the sampling table. Start the automatic sampling machine (see Table of Materials), and open the air control valve.</li>
	<li>Open the visionCATS software (see Table of Materials). Click New &#62; New Folder (named Rhodiola crenulata sample test) &#62; OK. Click New Method (name Rhodiola crenulata sample test) &#62; OK &#62; ATS 4.</li>
	<li>Click Finish Step Definition. Click Track Assignment to edit the description (gallic acid, salidroside, tyrosol, and Rhodiola crenulata sample).</li>
	<li>Click HPTLC Steps. Set the thin layer parameters (5 cm x 10 cm). Select Application Type (band), set the parameters (Table 1), and click the OK button.</li>
	<li>Open Filling/Rinsing Quality. Check Fill only Programmed Volume. Set the vial bottom level (mm) to 0.5, and click the OK button.</li>
	<li>Click Execute Method.</li>
	<li>Click the Track Assignment button, check Center, and set the parameters (Table 2).</li>
	<li>Click the Continue button for automatic sampling.</li>
	<li>Turn off the automatic sampling machine and air valve. Remove the silicone sheet from the automatic sampling machine.</li>
	<li>Put the silicone sheet into the other side of the double tank chromatography cylinder in step 6.1, cover the upper cylinder head, and pre-saturate for 20 min.</li>
	<li>Gently clamp the upper end of the thin layer plate with a tweezer, quickly put the silicone sheet into the development agent, and cover the upper cylinder head. 
	NOTE: Take out the silicone sheet when the unfolding front edge is 0.5-1.0 cm away from the upper end of the thin layer plate.</li>
	<li>After the organic solvent evaporates, spray the chromogenic solution onto the surface of the silicone sheet at room temperature to obtain chromogenic results. 
	NOTE: The chromogenic solution is an aqueous solution containing 2% FeCl3 and 1% K3[Fe(CN)6].</li>
</ol>

Microscopic Characterization and TLC-Based Identification of Rhodiola crenulate

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The authors have nothing to disclose.

There are more than 90 species of Rhodiola plants in the world, and more than 60% of all species are found in China, common ones including Rhodiola crenulata, Rhodiola rosea, Rhodiolas achalinensis, and Rhodiola algida17. Rhodiola crenulata, recorded in the first part of the Chinese Pharmacopoeia (2020), is a traditional Tibetan medicine grown at high altitudes. The market demand for Rhodiola crenulata is increasing yearly, so ensuring the correct use o...

Herbal medicine has a long history and rich application experience in China, and it was the first systematic recording in Shennong&#39;s herbal classic1. The discovery of artemisinin applied to malaria promoted the development of herbal medicine into a new stage1. The use of modern scientific technology to uncover the exact mechanism of herbal medicine increases the utilization rate and demand for herbal medicine, opening a new international market for it2,3,4. However, this has led to a series of negative effects. Non-professionals have a vague understanding of the characteristics of herbal medicine, which makes the use of herbal medicine face a huge safety risk5.
Rhodiola crenulata, one of the plants of the Rhodiola species, is mainly distributed in Tibet, northwest Yunnan, and western Sichuan of China (Figure 1)6,7. Rhodiola crenulata comprises salidroside, tyrosol, gallic acid, and other compounds for the treatment of hypoxic-related diseases through the function of &#34;invigorating qi and promoting blood circulation, clearing pulse and calming asthma&#34;8,9,10,11. Field investigation shows that Rhodiola crenulata can be found in the alpine talus zones, gully slopes, and rock crevices at an altitude of 4,000-5,600 m. Its growing environment is cold, full of sunshine and intense radiation, and it belongs to the alpine meadow ecosystem. Rhodiola crenulata can be distributed in lamellar and point-like populations according to the growth terrain, and gene flow can be carried out through cross-pollination.
The pollen abortion of the genus Rhodiola, illegality excavation, and degenerated ecological environment make Rhodiola crenulata an endangered species6,12. In view of the high medicinal value of Rhodiola crenulata, counterfeit products are expected to flow into the market. This article presents the habitat of Rhodiola crenulata and some convenient laboratory identification methods. Firstly, we observed the growth environment of Rhodiola crenulata and its medicinal properties. Secondly, the microstructure of medicinal powder was observed by microscope. The last step is the key point. The representative components of Rhodiola crenulata were separated and identified according to the different adsorption or dissolution properties of these components in a certain substance. DNA-based authentication or metabolomics analysis methods of medicinal plants are complicated and expensive13. These basic, convenient, and economical methods can quickly identify Rhodiola crenulata.

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	<tbody>
		<tr>
			<td>0.22 &#956;m millipore filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic sampling machine</td>
			<td>CAMAG</td>
			<td>ATS 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate</td>
			<td>Fuzhou Brunei Technology Co., Ltd</td>
			<td>ST1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatographic&#160;sample&#160;bottles</td>
			<td>Zhejiang ALWSCI Technology Co., Ltd</td>
			<td>C0000008</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical flask</td>
			<td>Sichuan Shubo Co., Ltd</td>
			<td>1121</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Citotest Labware Manufacturing Co., Ltd</td>
			<td>10211818c</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting needle</td>
			<td>Shanghai Bingyu Fluid Technology Co., Ltd</td>
			<td>BY-5026</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>SHIMADZU</td>
			<td>ATX124</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>2022120901</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>2021110801</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallic acid</td>
			<td>Chengdu Herbpurify Co., Ltd</td>
			<td>M-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10010618</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed&#160; crusher</td>
			<td>Beijing Zhongxingweiye Instrument Co., Ltd</td>
			<td>FW-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>20230108</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Chongqing Oprec Nistrument Co.,&#160; Ltd</td>
			<td>B203</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Citotest Labware Manufacturing Co., Ltd</td>
			<td>7105P-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Shanghai Yuejin Medical Equipment Co., Ltd</td>
			<td>DHG-8145</td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmacopoeia sieve</td>
			<td>Hangzhou Xingrun sieve factory</td>
			<td>572423281330</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Changde BKMAM Biotechnology Co., Ltd</td>
			<td>120302008</td>
			<td></td>
		</tr>
		<tr>
			<td>Salidroside</td>
			<td>Chengdu Herbpurify Co., Ltd</td>
			<td>H-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Saturate tank&#160;</td>
			<td>Yancheng Liegu Technology Co., Ltd</td>
			<td>10*20 P-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel plate</td>
			<td>Yantai Jiangyou Silica Gel Development&#160; Co., Ltd</td>
			<td>HSG20211227</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloromethane</td>
			<td>Chengdu Kelong Chemical Co., Ltd</td>
			<td>20221013-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer&#160;</td>
			<td>Changde BKMAM Biotechnology Co., Ltd</td>
			<td>130302027</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosol</td>
			<td>Chengdu Herbpurify Co., Ltd</td>
			<td>L-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound equipment</td>
			<td>Ningbo Xinyi Ultrasonic Equipment Co., Ltd</td>
			<td>SB-8200DTS</td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric pipet</td>
			<td>Changde BKMAM Biotechnology Co., Ltd</td>
			<td>120301006</td>
			<td></td>
		</tr>
	</tbody>
</table>

field collection and laboratory routine identification of rhodiola crenulata

The identification of medicinal materials is the premise and guarantee of drug safety. The majority of scientific researchers are bound to favor the simple, fast, effective, and inexpensive identification process of herbals. Rhodiola crenulata is a traditional Tibetan medicine grown at high altitudes, mainly distributed in Tibet, Yunnan, and Sichuan regions of China. Rhodiola crenulate possesses multiple bioactivities, such as anti-inflammatory, anti-hypoxia, and antioxidant properties, and has great potential for development. With the increasing market demand and a rapid decrease in resource content, a large number of confused products of Rhodiola crenulata have been troubling people. Therefore, this protocol introduces a standard process for the identification of Rhodiola crenulata in the field combined with routine laboratory testing. The combination of habitat, microscopic features, and thin-layer chromatography will undoubtedly identify Rhodiola crenulata quickly, efficiently, and economically, contributing to the continuous development of Tibetan medicine and the quality control of medicinal materials.

This experimental protocol describes identifying and collecting Rhodiola crenulate in the field. Rhodiola crenulate tends to live in the alpine talus zones, gully slopes, and rock crevices at high altitudes. The habitat, whole plant, flower, and leaves of Rhodiola crenulate can be shown in Figure 2. Rhodiola crenulate has a reddish-brown rhizome (Figure 3A). A representative image of the medicinal powder is shown in <strong cl...

Watch this Scientific Journal Video about Field Collection and Laboratory Routine Identification of Rhodiola crenulata at JoVE.com

Author Spotlight: Developing Identification Methods for Rhodiola crenulata and Investigating Its Resource Distribution and Pharmacological Effects

Here, we describe the identification of Rhodiola Crenulata from habitat, plant morphology, medicinal properties, microscopic features, and thin-layer chromatography.

Field Collection and Laboratory Routine Identification of Rhodiola crenulata

The identification of medicinal materials is the premise and guarantee of drug safety. The majority of scientific researchers are bound to favor the ...

The scope of our research is centered on conducting a resource survey of ethnic medicine. Specifically, this study aims to develop a rapid and convenient methods for the identification of Rhodiola crenulata. The protocol we offer presents a cost-effective, straightforward, and rapid method for identifying various medicinal herbal materials.Our laboratory will concentrate its future research endeavors on investigation two primary areas, the distribution of research associated with Rhodiola crenulata and the pharmacological impacts of this species.

field-collection-laboratory-routine-identification-rhodiola

Research

JoVE Journal

Medicine

482 Views.  Chengdu University of Traditional Chinese Medicine. The scope of our research is centered on conducting a resource survey of ethnic medicine. Specifically, this study aims to develop a rapid and convenient methods for the identification of Rhodiola crenulata. The protocol we offer presents a cost-effective, straightforward, and rapid method for identifying various medicinal herbal materials.Our laboratory will concentrate its future research endeavors on investigation two primary areas, the distribution of research associated with Rhodi...

Video: Author Spotlight: Developing Identification Methods for Rhodiola crenulata and Investigating Its Resource Distribution and Pharmacological Effects

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Remote Laboratory Management: Respiratory Virus Diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

Learning Modern Laryngeal Surgery in a Dissection Laboratory

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is almost completely reserved to a few referral centers that deal with a large proportion of this pathology. Practice on human specimens is not always possible for ethical, economic, or availability reasons. The aim of this study is to provide a reproducible method for the organization of a laryngeal laboratory on ex vivo animal models where it is possible to approach, learn, and refine laryngeal techniques. Porcine and ovine larynges are ideal, affordable, models to simulate laryngeal surgery given their similarity to the human larynx in their anatomical layout and tissue composition. Herein, the surgical steps of transoral laser surgery, open partial horizontal laryngectomy, and total laryngectomy are reported. The merging of endoscopic and exoscopic views guarantees an inside-out perspective, which is vital for the comprehension of the complex laryngeal anatomy. The method was successfully adopted during three sessions of a dissection course &#34;Lary-Gym&#34;. Further perspectives on robotic surgical training are described.

The purpose of this paper is to illustrate how to organize a reproducible laboratory for laryngeal surgery on affordable and closely similar animal laryngeal models in order to improve anatomical and surgical knowledge and skills.

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is ...

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, establishing a method to determine the multi-component in vitro dissolution of RG is of great significance for quality control. This study uses the second paddle method of the fourth general rule 0931 from the Chinese Pharmacopoeia (2020 edition), compliant with apparatus 2 of the United States Pharmacopeia (USP). The dissolution apparatus was set to a rotation speed of 100 rpm with ultrapure water as the dissolution medium. A sample volume of 1 mL was collected at each timepoint. Furthermore, the cumulative dissolution of gallic acid, salidroside, and ethyl gallic acid in RG at different time points was determined by high-performance liquid chromatography (HPLC). Finally, the dissolution curves were drawn, and the curves were fitted to the GompertzMod, the Gompertz, the Logistic, and the Weibull equations. The results showed that the cumulative dissolution of gallic acid in RG was over 80% at 1 min, the cumulative dissolution of salidroside and ethyl gallic acid was over 65% at 5 min, and the cumulative dissolution of each index component decreased after 30 min. The curve fitting demonstrated that the GompertzMod equation was the best-fitting model for each index component of RG. In conclusion, the dissolution test method described in this protocol is simple, accurate, and reliable. It can characterize the dissolution behavior of the index components in RG in vitro, which provides a methodological reference for quality control of RG and quality evaluation of other ethnic compounds.

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

Here, we test the dissolution of Rhodiola granules (RG) in vitro, draw dissolution curves of salidroside, gallic acid, and ethyl gallate in ultrapure water, and fit the curves to different mathematical models. This protocol provides information and guidance for in vivo bioequivalence and in vivo-in vitro correlation studies of RG.

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, ...

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like diabetes and obesity, and chronic inflammatory diseases like atherosclerosis, cancer, and autoimmune diseases. Upon vascular injury, usually the coagulation system, platelets, and endothelium act in an orchestrated manner to prevent bleeding by forming a clot at the site of the injury. Abnormalities in this process lead to either excessive bleeding or uncontrolled thrombosis/insufficient antithrombotic activity, which translates into vessel occlusion and its sequelae. The FeCl3-induced carotid injury model is a valuable tool in probing how thrombosis initiates and progresses in vivo. This model involves endothelial damage/denudation and subsequent clot formation at the injured site. It provides a highly sensitive, quantitative assay to monitor vascular damage and clot formation in response to different degrees of vascular damage. Once optimized, this standard technique can be used to study the molecular mechanisms underlying thrombosis, as well as the ultrastructural changes in platelets in a growing thrombus. This assay is also useful to study the efficacy of antithrombotic and antiplatelet agents. This article explains how to initiate and monitor FeCl3-induced arterial thrombosis and how to collect samples for analysis by electron microscopy.

The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis.

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like ...

A Protocol to Isolate, Culture, and Identify Human Limbal Niche Cells

Isolation and Identification of Limbal Niche Cells

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 &#176;C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal&#160;epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 &#176;C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFR&#946;). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFR&#946;, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were&#160;68%, 99%, 20%, and 3% in&#160;BMMSCs. The standard process for LNC&#160;isolation and identification could provide&#160;a reliable laboratory basis for the widespread use of LNCs.

Author Spotlight: Standardizing Limbal Niche Cell (LNC) Isolation and Characterization to Support Widespread LNC Research 

Here, we present a protocol to isolate and identify the human limbal niche cells.

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...