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Isolation of the Brain Secretome from Ex Vivo Brain Slice Cultures
In This Article
Summary
The brain secretome plays a pivotal role in the development and normal functioning of the central nervous system. In this article, we provide a detailed protocol for isolation of the brain secretome from ex vivo brain slice cultures.
Abstract
The brain secretome consists of proteins either actively secreted or shed from the cell surface by proteolytic cleavage in the extracellular matrix of the nervous system. These proteins include growth factor receptors and transmembrane proteins, among others, covering a broad spectrum of roles in the development and normal functioning of the central nervous system. The current procedure to extract the secretome from cerebrospinal fluid is complicated and time-consuming, and it is difficult to isolate these proteins from experimental animal brains. In this study, we present a novel protocol for isolating the brain secretome from mouse brain slice cultures. First, the brains were isolated, sliced, and cultured ex vivo. The culture medium was then filtered and concentrated for isolating proteins by centrifugation after a few days. Finally, the isolated proteins were resolved using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently probed for purity characterization by western blot. This isolation procedure of the brain secretome from ex vivo brain slice cultures can be used to investigate the effects of the secretome on a variety of neurodevelopmental diseases, such as autism spectrum disorders.
Introduction
The extracellular matrix of the nervous system consists of proteins that are either actively secreted or shed from the cell surface, called the "secretome". The brain secretome contains proteins such as growth factor receptors and transmembrane proteins1, which encompass a broad spectrum of roles in the development and normal functioning of the central nervous system. A large number of proteins secreted by the glial cells, including microglia and astrocytes in particular, reflect a wide variety of glial conditions, including neuroinflammation in the central nervous system2. Protein secretome has been demonstrated....
Protocol
This protocol was approved and follows the animal care guidelines set by the Southern University of Science and Technology Animal Care and Use Committee (SUSTech-JY202112006). Adult male and female C57BL/6J mice (6-8 weeks old, 22-30 g) were used in this study. Mice were housed at 22-25 °C on a circadian cycle of 12 h of light and 12 h of darkness, with ad libitum access to food and water. The steps of the protocol are listed as follows.
1. Isolation of brain slices
Representative Results
To quantify the secretion of extracellular proteins in brain slices, we examined protein concentrations of the brain slice samples and conditioned medium samples through BCA assay experiments. Brain slice samples and conditioned medium samples all had high protein concentrations (Table 1 and Figure 2). We found that a large number of extracellular proteins were secreted into the medium, and the average protein concentrations in the conditioned medium were ca.......
Discussion
The brain secretome refers to the collection of signaling molecules, known as neurosecretory or neuropeptide products, that are released by neurons or glial cells into the extracellular environment. The brain secretome holds critical functions in many biological and physiological processes in the nervous system17,18. Understanding the brain secretome and its functions is important for gaining insight into the mechanisms underlying brain function and disorders. Mo.......
Acknowledgements
This research was supported by Shenzhen Clinical Research Center for Mental Disorders (20210617155253001), Shenzhen Fund for Guangdong Provincial High-level Clinical Key Specialties (SZGSPO13), Shenzhen Science and Technology Innovation Committee (JCYJ20200109150700942), Key-Area Research and Development Program of Guang Dong Province (2019B030335001), and Shenzhen Key Medical Discipline Construction Fund (SZXK042) We are grateful for the assistance of Guangdong Provincial Key Laboratory of Advanced Biomaterials.
....Materials
| Name | Company | Catalog Number | Comments |
| 0.22 μm Non-Sterile Millex Syringe Filters with PES Membrane | Millipore | SLGPR33RB | |
| 1,4-Dithio-DL-threitol (DTT) | Merck (Sigma Aldrich) | DTT-RO | |
| 6-well plates | CORNING | 3516 | |
| Actin antibody | Cell Signaling Technology | Â #3700 | |
| APP Antibody | Cell Signaling Technology | 2452 | |
| BioRad Mini-Protean TGX precast gels | BioRad | #1610185 | |
| Bovine serum albumin (BSA) | Merck (Sigma Aldrich) | 10735094001 | |
| Bromophenol Blue | Merck (Sigma Aldrich) | B0126 | |
| Brush pen | Deli | 1.00008E+11 | |
| CaCl2 | Merck (Sigma Aldrich) | C1016 | |
| CF3COOH | Merck (Sigma Aldrich) | 302031 | |
| CH3CN | Merck (Sigma Aldrich) | 34851 | |
| Clusterin antibody | Cell Signaling Technology | #42143 | |
| D-Glucose | Merck (Sigma Aldrich) | G8270 | |
| Easy-nLC1200 | Thermo Fisher Scientific | Easy-nLC1200 | |
| Filter paper | ROHU | ROHU-DLLZ00105 | |
| GAPDH antibody | Cell Signaling Technology | # 5174S | |
| Glycerol | Merck (Sigma Aldrich) | G5516-100ML | |
| Hanks' Balanced Salt Solution (HBSS) | Invitrogen (Gibco) | 14175095 | |
| Human/Rat NLGN2 Antibody | R&D Systems | AF5645 | |
| ICH2CONH2 | Merck (Sigma Aldrich) | I6125 | |
| Immun-Blot PVDF membranes | BIO-RAD | #1620177 | |
| KCl | Merck (Sigma Aldrich) | P3911 | |
| L-Ascorbic Acid | Merck (Sigma Aldrich) | A92902 | |
| Map2 antibody | Cell Signaling Technology | # 4542S | |
| MgSO4 | Merck (Sigma Aldrich) | M7506 | |
| m-IgGκ BP-HRP | SANTA CRUZ BIOTECHNOLOGY | sc-516102 | |
| Millicell-CM 0.4 μm | Millipore | PIHP03050 | |
| Millipore AmiconUltra-0.5ML | Millipore | UFC5010 | |
| mouse anti-rabbit IgG-HRP | SANTA CRUZ BIOTECHNOLOGY | sc-2357 | |
| NaH2PO4 | Merck (Sigma Aldrich) | S0751 | |
| NaHCO3 | Merck (Sigma Aldrich) | S5761 | |
| NH4HCO3 | Merck (Sigma Aldrich) | A6141 | |
| Parenzyme | Thermo Fisher Scientific | R001100 | |
| Penicillin-Streptomycin Solution(P/S) | Invitrogen (Gibco) | 15070063 | |
| Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | 23225 | |
| QEactive | Thermo Fisher Scientific | IQLAAEGAAPFALGMAZR | |
| Rabbit Anti-Goat IgG (H&L)-HRP Conjugated | Easybio | BE0103-100 | |
| Razor blade | BFYING EAGLE | HX-L146-1H | |
| Sodium chloride NaCl | Merck (Sigma Aldrich) | S6150 | |
| Sodium dodecylsulfate (SDS) | Merck (Sigma Aldrich) | V900859 | |
| Sodium Pyruvate | Merck (Sigma Aldrich) | P2256 | |
| SpeedVac SRF110 Refrigerated Vacuum Concentrator (German) | Thermo Fisher Scientific | SRF110P2-115 | |
| Sucrose | Merck (Sigma Aldrich) | G8270 | |
| Tissue grinder | SCIENTZ | SCIENTZ-48 | |
| Tris (Hydroxymethyl) | Merck (Sigma Aldrich) | TRIS-RO | |
| TritonX-100 | Merck (Sigma Aldrich) | T8787 | |
| Tween-20 | Merck (Sigma Aldrich) | P1379 | |
| Vibratory slicer | Leica | Leica VT 1000S |
References
- MartÃn-de-Saavedra, M. D., et al. Shed CNTNAP2 ectodomain is detectable in CSF and regulates Ca2+ homeostasis and network synchrony via PMCA2/ATP2B2. Neuron. 110 (4), 627-643 (2022).
- Jha, M. K., et al.
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