This work was supported by the National&#160;Natural Science Foundation of China (82370281, 81870222), the Natural Science&#160;Foundation of Guangdong Province (2022A1515012103), and the Science, the&#160;Technology Development Special Fund Competitive Allocation Project of Zhanjiang City&#160;(2021A05086), and the Startup Foundation&#160;from the Second Affiliated Hospital of&#160;Guangdong Medical University (23H03).

The samples were obtained from mice, with ethical approval granted by the Ethics Committee of Guangdong Medical University. Detailed descriptions of materials, equipment, and software used in this protocol are provided in the Table of Materials. The details of the preparation prior to extraction are illustrated in Figure 1, while the process of Spleen-Exo extraction and enrichment is described in Figure 2.
1. Preparation
<ol>
	<li>Pre-cool high-speed and ultra-high-speed centrifuges to 4 &#176;C and set the temperature of a desktop shaker to 37 &#176;C.</li>
	<li>Prepare cell culture dishes (<img alt="Equation 1" src="/files/ftp_upload/67234/67234eq01.jpg" style="margin: auto;" />100 mm), 50 mL sterile centrifuge tubes, ice, and sterile cell strainer (<img alt="Equation 1" src="/files/ftp_upload/67234/67234eq01.jpg" style="margin: auto;" />70 &#181;m) as shown in Figure 1A, B.</li>
	<li>Sterilize scissors and forceps using 75% alcohol spray, followed by high-temperature sterilization. These tools are shown in Figure 1C.</li>
	<li>Place the spleen tissue in a sterile <img alt="Equation 1" src="/files/ftp_upload/67234/67234eq01.jpg" style="margin: auto;" />100 mm Petri dish on ice, and wash off the surface blood with 1x iced sterile phosphate-buffered saline (PBS). Dry the spleen tissues with sterile gauze and weigh them after drying. 
	NOTE: If the tissue is frozen, thaw it in a dish for 15 min on ice before proceeding.</li>
	<li>Moisturize the cell strainer (<img alt="Equation 1" src="/files/ftp_upload/67234/67234eq01.jpg" style="margin: auto;" />70 &#181;m) by dropping 1 mL of sterile PBS into the strainer using a sterile transfer pipet.</li>
	<li>Prepare the digestive buffer (0.1% type I collagenase) using 1x PBS with a vortex mixer. 
	NOTE: Use 10 mL of digestive buffer for every 100 mg of spleen tissue. The collagenase concentration was chosen in this study based on the product instructions. For other tissue types, researchers may need to adjust these parameters based on tissue density and composition. Preliminary experiments with varying concentrations and digestion times are recommended to determine the optimal conditions for each tissue type.</li>
</ol>
2. Tissue digestion
<ol>
	<li>Chop the tissue into uniform small pieces with scissors in a culture dish on ice (Figure 3A) and transfer to a 50 mL centrifuge tube with a sterile transfer pipet.</li>
	<li>Add digestion buffer to the tube according to the spleen tissue weight as indicated above (Figure 3B), then shake the tube with an angle of 45&#176; on a horizontal shaker at 37 &#176;C. Monitor the digestion progress and stop the digestion when tissue chunks have dispersed, and most have lost their shape. Digestion time should vary between tissues. Under the experimental conditions in this study, the digestion time is approximately 30 min.</li>
	<li>Transfer the digested mixture to a cell strainer (&#1060;70 &#181;m) at room temperature (RT) to remove fibrous and larger tissue debris. 
	NOTE: Complete filtration immediately after digestion.</li>
	<li>Collect the filtrate in a new 50 mL centrifuge tube.</li>
</ol>
3. Exosome isolation by differential centrifugation
<ol>
	<li>Centrifuge filtrates at 500 x g for 10 min at 4 &#176;C. A pellet containing residual cells, cell debris, and nuclei can be seen after centrifugation, as shown in Figure 3C.</li>
	<li>Transfer the supernatant to a new tube and centrifuge at 3000 x g for 20 min at 4 &#176;C. A pellet containing apoptotic bodies and microsomes can be seen after centrifugation, as shown in Figure 3D.</li>
	<li>Transfer the supernatant to a new tube and centrifuge at 10,000 x g for 30 min at 4 &#176;C. Pellet containing microvesicles and plasma membrane can be seen after centrifugation, as shown in Figure 3E.</li>
	<li>Ultracentrifuge the resulting supernatant at 120,000 x g for 2 h at 4 &#176;C. A pellet can be seen at the bottom of the tube after centrifugation, as shown in Figure 3F.</li>
	<li>Discard the supernatant, wash the pellet with PBS, and ultracentrifuge again at 120,000 x g for 2 h at 4 &#176;C to obtain exosomes. A pellet can be seen at the bottom of the tube after centrifugation, as shown in Figure 3G.</li>
	<li>Dissolve the isolated exosomes with sterile PBS (200 &#181;L per spleen) and store the isolated exosomes in a new 2 mL centrifuge tube at -80 &#176;C.</li>
</ol>
4.&#160;Exosome characterization by transmission electron microscopy (TEM)
NOTE: TEM was used to determine whether the extracted Exo displayed vesicle characteristics. The Exo samples identified by electron microscopy must be fresh samples or stored briefly at 4 &#176;C for a short time.
<ol>
	<li>Fix exosomes (2-6 &#181;g/&#181;L) with 2% electron microscopy (EM)-grade paraformaldehyde at RT for 2 h.</li>
	<li>Place 10 &#181;L of exosome solution on a copper mesh, incubate at RT for 10 min, rinse with sterile distilled water, and blot excess liquid with absorbent paper.</li>
	<li>Stain the copper mesh with 2% uranyl acetate for 1 min, blot excess fluid with filter paper, and dry under an incandescent lamp for 2 min.</li>
	<li>Observe the prepared sample under a transmission electron microscope at 80 kV.</li>
</ol>
5. Size distribution and particle concentration measurement of exosomes
<ol>
	<li>Load the standard polystyrene nanoparticles (250 nm) to the nano-flow cytometer.</li>
	<li>Measure the protein concentration of Exo samples using a BCA protein assay kit. Dilute Exo sample with PBS according to BCA Protein Assay results (dilute the exosomes to 1-10 ng/&#181;L), then load the Exo samples to the nano-flow to measure the side scatter intensity (SSI).</li>
	<li>Calculate Exo concentration according to the ratio of SSI to particle concentration in the standard polystyrene nanoparticles. 
	NOTE: For size measurement, a standard curve is generated by loading standard silica nanoparticles with gradient sizes (68 nm, 91 nm, 113 nm, 155 nm) to the nano-flow cytometer. The loading of the Exo sample follows this. The size distribution of Exo was calculated according to the standard cure.</li>
</ol>
6. Lysis and immunoblot confirmation of exosome proteins
<ol>
	<li>Add 100 &#181;L of radio immunoprecipitation assay (RIPA) lysis buffer to the exosome pellet for protein analysis.</li>
	<li>Vortex vigorously, cover with parafilm, rock for 20 min at RT, then vortex again.</li>
	<li>Centrifuge briefly at 1000 x g for 30-60 s to collect the sample, transfer to a new 1.5 mL microcentrifuge tube, and store at -20 &#176;C to -80 &#176;C until analysis.</li>
	<li>For immunoblot, mix samples with 5x loading buffer to achieve a 1x concentration and prepare for gel electrophoresis.</li>
	<li>If whole-tissue homogenate controls are desired, use a strong lysis buffer with a protease inhibitor to lyse the tissue, as detailed above. Then, centrifuge the samples at 10,000 x g for 10 min and discard the remaining extracellular matrix, whole cells, or intact cellular debris.</li>
	<li>Boil the sample buffer containing exosomes or tissue homogenate at 95 &#176;C for 5-10 min. Load equal protein per lane into a 10% SDS-PAGE gel. Load equal protein of tissue homogenate as control.</li>
	<li>Proceed with gel electrophoresis under a voltage of 80 V for approximately 2 h using the conventional electrophoresis buffer. Perform western blot analysis to confirm Exo protein expression in the purified lysates and compare relative Exo abundance in fractions.</li>
</ol>

Refining Techniques for Murine Spleen-Derived Exosome Isolation

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R., Hoenderop, J. G. J., Rigalli, J. 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The authors declare that they have no competing interests.

Recent research has reported that spleen-derived exosomes (Spleen-Exo) play a critical role in the treatment of gastric cancer12. To further elucidate the functions of Spleen-Exo, it is necessary to establish a reproducible and optimal method for their extraction from spleen tissue. While protocols exist for isolating exosomes from brain and liver cancer13,14, methods for other tissues remain underdeveloped and require further validation. In this study, we have outlined a practical method for isolating Exo from spleen tissue. 
Over the past years, various methods have been used to extract Exo in scientific research and applications, including ultracentrifugation, density gradient centrifugation, magnetic bead immunoadsorption, ultrafiltration, and polymer precipitation15. Ultracentrifugation remains the gold standard method for extracting Exo from various sources. Previous studies have focused on disrupting tissue structures to obtain higher concentrations of Exo, which consists of intracellular and extracellular sources of Exo16. Collagenase minimizes the impact on the integrity of the cell membrane, ensuring the purity of Ti-Exo3. Collagenase has been used to extract extracellular vesicles from human metastatic melanoma, adipose, colon cancer, and liver cancer tissues5,10,13,17. However, the concentration and digestion time of collagenase vary among different tissues. 
We established a specific protocol for isolating Spleen-Exo using Type I collagenase for digestion, followed by filtration through differential ultracentrifugation, to yield high-quality Spleen-Exo. Ultracentrifugation can effectively remove residual cells, large vesicles, cell debris, and apoptotic bodies from the samples16. Crescitelli et al. employed collagenase D and gradient ultracentrifugation to isolate small extracellular vesicles with a mean size of 75 nm in diameter5. Compared with their protocol, our protocol used type I collagenase and an additional ultracentrifugation step at 120,000 x g to achieve optimal purity of exosomes. In contrast to previous reports using tissue homogenization to obtain tissue suspension14,18, our approach preserves the membrane integrity of cells in tissues. In addition, Vella et al. applied density gradient centrifugation (a triple sucrose gradient) to generate three groups of particles, which required extra time for the operation10. This approach produced large vesicles with a size over 200 nm, a heterogeneous population of vesicles with sizes ranging from 50 nm to 200 nm, and small vesicles around 20 nm in size. The protocol described in this study protocol involves a series of ultracentrifugation steps to methodically eliminate residual cells, cell debris, apoptotic bodies, and larger vesicles, thus enhancing the purity of the Exo obtained. We emphasize careful handling during supernatant collection to avoid disturbing the sedimented pellets. 
We assess the morphology and purity of the isolated Spleen-Exo through TEM and nanoparticle tracking analysis. Both assays revealed that the spleen-derived particles isolated by this protocol exhibited a lipid bilayer and cup-shaped vesicles with sizes ranging from 30 nm to 150 nm, which are the typical characteristics of exosomes19. CD9 and TSG101 are exosome markers19, and GM130, a Golgi protein, is used as a negative marker for exosome identification20. The results showed that Spleen-Exo were positive for CD9 and TSG101, while GM130 was not detected, confirming the successful isolation of high-quality, tissue-derived Exo from spleens.
In achieving high-quality Ti-Exo, selecting the appropriate enzyme type, concentration, and digestion duration, as well as proper tissue storage, is essential. We observed no significant difference in protein concentrations between Exo derived from fresh versus frozen spleen tissues (data not shown), indicating the feasibility of processing large quantities of frozen tissues to reduce the workflow. Isolating Ti-Exo requires a long operating time for ultracentrifuge, making it impractical to complete the whole protocol within an appropriate period when using fresh tissues in clinical applications. Thus, using frozen tissues for exosome isolation would be a better option. Thawing at 37 &#176;C is not suitable for frozen tissues due to incomplete penetration of cryoprotective agents in tissues. It was reported that when performing exosome isolation using frozen tissues thawing on ice, the yield of exosomes showed no significance in content compared with fresh tissues21. Thus, we thawed the frozen spleen tissues on ice to perform exosome isolation when using the storage samples at -80 &#176;C. 
However, the protocol described in this study has several limitations. The extensive steps involved in extraction and identification are time-consuming. Given the ultimate goal of applying Exo research to clinical disease treatment, faster and more cost-effective methods are becoming increasingly critical. Additionally, while the results are based on mouse tissue samples, the applicability to human tissues remains to be determined.
In conclusion, the protocol provides a method for extracting highly pure Exo from spleen tissue, suitable for detailed downstream analysis. Tissue-derived Exo reflects the microenvironment of their origin more accurately and holds significant potential for elucidating physiological and pathological processes in living organisms. Future studies could adapt this protocol to human tissues, enabling the extraction of clinically relevant exosomes for diagnostic and therapeutic purposes. This technique could be particularly useful in studying the role of exosomes in immune responses, given the spleen's critical function in the immune system. Moreover, the protocol could be modified to isolate exosomes from other tissues, potentially aiding in the development of tissue-specific biomarkers and targeted therapies. By improving our understanding of exosome biology, this protocol can contribute to advancements in precision medicine and the development of novel therapeutic strategies.

Exosomes (Exo) are a subgroup of extracellular vesicles (EVs), ranging from 30 to 150 nm in size, encapsulating a diverse array of biomolecules, including proteins, nucleic acids, and lipids1. These biomolecules are derived from the parental cells and are released into the extracellular space. Exo facilitate biomolecular information exchange between cells and their surrounding microenvironment, playing roles in both prokaryotic and eukaryotic systems2. The characteristics of exosomes, such as content, size, membrane components, and cellular origin, are highly variable and influenced by the originating cell type, cellular state, and environmental conditions.
Exosomes are commonly classified into three categories based on their source: cell culture-derived, body fluid-derived, and tissue-derived (Ti-Exo). While cell culture-derived exosomes have been extensively studied due to their accessibility and consistent yield, their use is limited by potential alterations in cell characteristics after prolonged culture, which may misrepresent their biological functions3,4,5. Moreover, most cell cultures are maintained in two-dimensional environments that do not mimic the complex in vivo intercellular interactions, potentially impacting data interpretation6. Conversely, exosomes isolated from biological fluids offer a minimally invasive option to monitor disease progression in real time7. However, these samples often contain a mixture of exosomes from various origins, complicating the accurate identification of their primary source8. Given these challenges, there is an increasing interest in Ti-Exo, which reside in the extracellular interstitium and are key mediators of intercellular signaling.
The spleen plays a crucial role in immune function and maintaining internal homeostasis. Despite existing protocols for isolating Ti-Exo from organs such as the brain, liver, and tumors, practical methods for spleen-derived exosomes are limitedly reported9,10. This study aimed to establish a practical protocol for isolating exosomes from spleen tissue in mice modified from a previous report11. We detail a method involving collagenase digestion followed by ultracentrifugation, which minimizes the disruption of the cell membrane and ensures high purity and yield of spleen-derived Exo. The characteristics of isolated Exo using this established protocol were validated through electron microscopy (TEM), the nano-flow cytometer (Nano-FCM), and western blot, confirming the protocol&#39;s efficacy for further experimental research.

<table><tbody><tr><td>70 &amp;micro;m Cell Strainer</td><td>Biologix Group Co., Ltd.USA</td><td>15-1070</td><td></td></tr><tr><td>Anti CD9 antibody</td><td>Cell Signaling Technology, Inc (CST), USA</td><td>983275</td><td></td></tr><tr><td>Anti GM130 antibody</td><td>Proteintech Group, Inc.China&amp;nbsp;</td><td>66662-1-lg</td><td></td></tr><tr><td>Anti TSG101 antibody</td><td>Abcam Plc, UK</td><td>125011</td><td></td></tr><tr><td>Cell Culture Dish</td><td>Wuxi NEST Biotechnology Co.,Ltd, China</td><td>704001</td><td></td></tr><tr><td>Centrifuge tube</td><td>Wuxi NEST Biotechnology Co.,Ltd, China</td><td>788211</td><td></td></tr><tr><td>Collagenase Type I</td><td>Sigma-Aldrich Corp., USA</td><td>C2674</td><td></td></tr><tr><td>Desktop Thermostatic Shaker</td><td>Shanghai bluepard instruments Co., Ltd., China</td><td>THZ-100</td><td></td></tr><tr><td>Electrophoresis buffer</td><td>Wuhan Servicebio Technology Co., Ltd., China&amp;nbsp;</td><td>G2081-1L</td><td></td></tr><tr><td>Enhanced BCA Protein Assay Kit</td><td>Shanghai Beyotime Biotechnology Co., Ltd., China&amp;nbsp;</td><td>P0010S</td><td></td></tr><tr><td>Flow NanoAnalyzer</td><td>NanoFCM Inc., China</td><td>N30E</td><td></td></tr><tr><td>Fluorescence/Chemiluminescence imaging system&amp;nbsp;</td><td>Guangzhou Biolight Biotechnology Co., Ltd., China</td><td>GelView 6000Plus</td><td></td></tr><tr><td>HRP Goat anti-Mouse IgG</td><td>Proteintech Group, Inc.China&amp;nbsp;</td><td>15014</td><td></td></tr><tr><td>HRP Goat anti-Rabbit IgG</td><td>Proteintech Group, Inc.China&amp;nbsp;</td><td>15015</td><td></td></tr><tr><td>microplate reader</td><td>Molecular Devices, USA</td><td>CMax Plus</td><td></td></tr><tr><td>Microscissors</td><td>Shanghai&amp;nbsp;Medical&amp;nbsp;Instruments (group)&amp;nbsp;Co., Ltd.,China</td><td>WA1010</td><td></td></tr><tr><td>Microscopic tweezers</td><td>Shanghai&amp;nbsp;Medical&amp;nbsp;Instruments (group)&amp;nbsp;Co., Ltd.,China</td><td>WA3010</td><td></td></tr><tr><td>Multifuge X1R Pro centrifuge</td><td>Thermo Fisher Scientific, USA</td><td>75009750</td><td></td></tr><tr><td>Ophthalmic scissors</td><td>Shanghai&amp;nbsp;Medical&amp;nbsp;Instruments (group)&amp;nbsp;Co., Ltd.,China</td><td>Y00030</td><td></td></tr><tr><td>Ophthalmic tweezers</td><td>Shanghai&amp;nbsp;Medical&amp;nbsp;Instruments (Group)&amp;nbsp;Co., Ltd., China&amp;nbsp;</td><td>JD1060</td><td></td></tr><tr><td>Optima XPN-100 Ultrafiltration centrifuge</td><td>Beckman Coulter, USA</td><td>A94469</td><td></td></tr><tr><td>phosphate buffered saline (PBS)</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd., China</td><td>P1003</td><td></td></tr><tr><td>RIPA lysis buffer (strong, without inhibitors)</td><td>Shanghai Beyotime Biotechnology Co., Ltd., China&amp;nbsp;</td><td>P0013K</td><td></td></tr><tr><td>Ruler</td><td>Deli&amp;nbsp;Manufacturing&amp;nbsp;Company, China</td><td></td><td></td></tr><tr><td>SDS-PAGE Sample Loading Buffer, 5x</td><td>Shanghai Beyotime Biotechnology Co., Ltd., China&amp;nbsp;</td><td>P0015</td><td></td></tr><tr><td>Transfer Pipet</td><td>Biologix Group Co., Ltd.USA</td><td>30-0238A1</td><td></td></tr><tr><td>Transmission Electron Microscope</td><td>Hitachi, Japan</td><td>H-7650</td><td></td></tr><tr><td>Ultracentrifugation tube</td><td>Beckman Coulter, USA</td><td>355618</td><td></td></tr><tr><td>Western Transfer Buffer&amp;nbsp;</td><td>Wuhan Servicebio Technology Co., Ltd., China&amp;nbsp;</td><td>G2028-1L</td><td></td></tr></tbody></table>

isolation and characterization of exosomes derived from mouse spleen tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

Isolation and purification of spleen-derived exosomes 
To isolate exosomes from mouse spleen tissue, we employed a combination of collagenase digestion and differential ultracentrifugation (Figure 2). The initial steps involved pre-treating the spleen tissue to remove surface blood, followed by digestion with Type I collagenase. This enzymatic treatment facilitated the breakdown of extracellular matrix components, thus liberating the exosomes while preserving their integrity. Post-digestion, a series of centrifugation steps were employed to sequentially remove cellular debris, apoptotic bodies, and larger vesicles, culminating in the ultracentrifugation of the supernatant at 120,000 x g to pellet the exosomes. Subsequent isolation procedures yielded PBS-soluble pellets enriched with spleen-derived Exo, as shown in Figure 3G.
TEM results 
The morphology of the isolated exosomes was examined using transmission electron microscopy, a gold-standard method to study the characterization of exosomes. TEM is commonly used to validate the presence of exosomes, evaluate the quality of exosomes, and observe exosomes&#39; morphology. The TEM images (Figure 4A) revealed the presence of cup-shaped vesicles, which are characteristic of exosomes. These vesicles displayed a clear lipid bilayer structure, confirming the successful isolation of exosomes from the spleen tissue.
Size distribution of Spleen-Exo 
The size distribution and concentration of the exosomes were determined using a nano-flow cytometer. The results (Figure 4B) indicated that the diameter of the isolated exosomes ranged from 30-150 nm, with a peak concentration at approximately 60 nm. The diameter of isolated particles was dominantly from 50-80 nm, with a mean particle concentration of 4.6 x 109 particles/mL. This size range is consistent with previously reported characteristics of exosomes, further validating the isolation protocol.
Western blot analysis 
To confirm the presence of exosomal markers, we performed a western blot analysis. The exosomal markers TSG101 and CD9 were detected in the Spleen-Exo samples, whereas GM130, a Golgi matrix protein used as a negative control, was not detected (Figure 4C). In comparison, the protein of these markers was expressed in mouse spleen tissues (Spleen-T). This indicates the high purity of the isolated exosomes, as they were devoid of cellular contaminants.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67234/67234fig01.jpg" /> 
Figure 1: Supply preparation. (A) 50 mL centrifuge tubes, culture dishes, ice, and surgery tools were placed in a biosafety cabinet. (B) A sterile cell strainer (<img alt="Equation 1" src="/files/ftp_upload/67234/67234eq01.jpg" style="margin: auto;" />70 &#181;m). (C) Sterilized surgery tools. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67234/67234fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67234/67234fig02.jpg" /> 
Figure 2: Spleen-Exo isolation procedure. Prepare spleen tissue and cut it into small pieces in iced PBS, followed by digestion with Type I collagenase and centrifugation as indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67234/67234fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67234/67234fig03.jpg" /> 
Figure 3: Representative images of Spleen-Exo isolation. (A) Small spleen blocks ready for subsequent digestion. (B) Spleen tissue blocks with digestion buffer. (C) Pellets after 500 x g centrifugation. (D) Pellets after 3,000 x g centrifugation. (E) Pellets after 10,000 x g centrifugation. (F) Pellets after 120,000 x g centrifugation. (G) Exosome pellets after 120,000 x g centrifugation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67234/67234fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67234/67234fig04.jpg" /> 
Figure 4: Identification of Spleen-Exo. (A) TEM results showing isolated particles as round or oval membranous vesicles with cup-shaped morphology. Scale bar = 200 nm and 50 nm, respectively. (B) Size distribution analysis by Flow Nano Analyzer showed that the diameter of Spleen-Exo ranged dominantly from 50 nm to 80 nm, with a mean particle concentration of 4.6 &#215; 109 particles/mL. (C) Western blot analysis showing expression of exosome-specific marker proteins (TSG101 and CD9) in Spleen-Exo, with negative expression of GM130. These markers were detectable in protein samples extracted from mouse spleens (Spleen-T group). The representative western blot images were shown (n = 6 tissues). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67234/67234fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

isolation-characterization-exosomes-derived-from-mouse-spleen

Nanyang Technological University

Research

JoVE Journal

Medicine

511 Views.  Guangdong Medical University. Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Video: Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Biology

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Immunology and Infection

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables&#160;the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model.&#160;P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal&#160;P. gingivalis infection.

Microvesicles shed from the plasma membrane act as cellular effectors. Spleen microvesicles (SMVs) are surrogate markers of pathophysiological conditions. In rats and mice, SMV content and properties characterize aging or inflammation and are altered by cytoprotective treatments, making them a valuable yet abundant monitoring tool for preclinical research.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular ...

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...