If you use alternative products, the quality and effectiveness of the item may be affected. Astrocyte Control of Zika Infection Is Independent of Interferon Type I and Type III Expression. WebNormal Human Astrocyte Cells A Cells, Media and Reagents Information Lonza Cat No Name Contain CC-2565 NHA Cryopreserved > 1x10e6 cells / Amp CC-3186 AGM BulletKit Astrocyte Basal Medium, AGM SingleQuots CC-3187 Astrocyte Basal Medium, 500 ml rhEGF-B 0.5 ml CC-4123 AGM SingleQuots Insulin, 1.25 ml Ascorbic Acid, 0.5 ml GA Indeed, our 3D iPSC-induced cortical-like neuron and astrocyte co-culture with GRN loss strikingly and consistently recapitulated human specific TDP-43 cell pathology reported in human FTD patient brain. WebAlthough astrocytes regulate neuronal activity across many time domains, their cell-autonomous circadian clocks exert a particular role in controlling longer-term oscillations of brain function: the maintenance of sleep states and Are you sure you don't want to sign up to get news from ATCC? During the brain metastasis process, tumor cells separated from the primary tumor enter the systemic circulation, stop at the branch of cerebral micro vessels, and then pass through the damaged BBB to reach the brain parenchyma [7]. In Vitro Cellular & Developmental Biology Animal presents peer-reviewed manuscripts dealing with in vitro cultivation of cells, tissues, organs, and tumors from invertebrates and vertebrates. WebCell Line Description. Effects of exosomes on premetastatic niche formation in tumors. Two hundred l BCA working solution was added to each hole and placed at 37C for 30minutes. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. Astrocytes; glutamate transporter; immortalized cells; invitro model; nucleoside transporter; organic cation/anion transporter. 500,000 HA (2nd passage) frozen in Basal Medium w/10% FBS, 10% DMSO, Cryovial frozen HA (882A-05), Growth Medium (821-500), Subculture Rgnt Kit (090K), Shipped in Gr Med, 3rd psg (flasks or plates). Slowly proliferating foci with several morphologies appeared 4 months after initiation of the cultures and became progressively enriched by cells with a homogeneous appearance. Nature. In the brain PMN formation process, the exosomes secreted by tumor cells play the role of former sentry since they reach the brain tissue ahead of circulating tumor cells (CTCs) and exert the function of compromising the BBB integrity to accelerate tumor cells to pass through BBB. Overall, our study provides a new theoretical basis for lung cancer brain metastasis. An increasing number of studies have shown that astrocytes play a key role in regulating the formation of myelin sheath, which is the process of wrapping axons in myelin sheath, thereby increasing the transmission speed of electrical pulses. To download a certificate of analysis for C8-D1A [Astrocyte type I clone] (CRL-2541), enter the lot number exactly as it appears on your product label or packing slip. After 3 times washing, the permeable solution was added and incubated for 10min. Ten l 2% uranium diacetate was dropped onto the copper mesh and the sample was negatively stained for 1min. Creative Biolabs is the very first company to provide a wide range of leading technologies and products in the neuroscience research field. Primary human astrocytes were transfected with the SV40 T antigen expression vectors, pSV3neo or p735.6, and cultures were established with an extended lifespan. All the human SV40-derived lines retained morphological features and growth properties of type 1 astrocytes. Thank you for your interest in Astrocyte Cell Lines. In vitro astrocyte models are valuable experimental tools in various astrocyte studies. PMC 2021;33(10):20402058.e10. ZERO BIAS - scores, article reviews, Lobb RJ, Lima LG, Mller A. Exosomes: key mediators of metastasis and premetastatic niche formation. PubMed And the differentially expressed proteins were associated with a variety of cell components. The control group was SVG P12 cells without exosomes treatment, and the experimental group was SVG P12 cells co-cultured with exosomes of H1299 cells for 24h. The differences in protein expression between the two groups were compared. Clonal permanent cell lines with astrocytic or microglial properties have been established from explant cultures of 8-day postnatal mouse cerebella after in vitro spontaneous transformation. The 96-well plates were placed in an incubator for 24hours and removed. It is well known that metastatic tumor cells require specific energy, nutrient, and oxygen patterns to compete with the in-situ cells at the site of metastases so that they could adapt to the local tissue microenvironment and establish metastatic colonies. Epub 2017 Nov 11. 2014. Computational Models of Neurons and Astrocytes in Studying Brain Dynamics in Health and Disease Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53% (w/v) EDTA solution to remove all traces of serum that contains trypsin inhibitor. Plant in January 1991 and continued by In Vitro Cellular & Developmental Biology. Your application may be in one of the following statuses: You can check the status of your application in your My Dashboard portal. As shown in Fig. You are now leaving atcc.org to securely complete your transaction on lgcstandards.com. A Results of KEGG pathway annotation of differentially expressed proteins in SVG P12 cells. Studies of brain cell function and physiology are hampered by the limited availability of immortal human brain-derived cell lines, as a result of the technical difficulties encountered in establishing immortal human cells in culture. Treatment of astrocytes with exosomes released from precursor B acute lymphoblastic leukemia embryo cells leads to the upregulation of vascular endothelial growth factor (VEGF), a vital factor that mediates leukemia infiltration into the central nervous system [15]. -, Neuropathol Appl Neurobiol. Once UTSW63 and UTSW71 cell lines were established, only low passage cells were used. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Accessibility Careers. 1994 Nov;63(5):1975-8 ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information. We welcome talented scientists and specialists in the neuroscience field to join us. Cell culture supernatant was centrifuged at 2000g for 30min to remove cells and debris. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The data of different groups were analyzed by unpaired t-test. Guo Y, Ji X, Liu J, et al. Article 2012;21(5):64254. One of these cultures gave rise to an immortal cell line, designated A735. WebClonal permanent cell lines with astrocytic or microglial properties have been established from explant cultures of 8-day postnatal mouse cerebella after in vitro spontaneous How to Select AAV Serotypes for Neurological Research? The astrocyte type II cloned cell line named C8-S is available as ATCC CRL-2535 and the astrocyte type III cloned cell line named C8-D30 is available as ATCC CRL-2534. The focus is on biological research, development, and applications of significance to science and society. Micromachines (Basel). Bresnick AR, Weber DJ, Zimmer DB. To further explore the influence of exosomes extracted from H1299 cells on SVG P12, we used flow cytometry to measure the apoptosis of SVG P12 cells treated with H1299 cell-derived exosomes (the concentration was 60g/ml) for 24h. The results showed that compared with the control group, the early and late apoptotic rates of the treatment group were significantly increased (Fig. Decontaminate the bottle with 70% alcohol in a sterile hood. Our data suggest that astrocytes in primary culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation. 2016;30(5):66881. 2 , 3 , 4 Keep up to date with our events, news, and more. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cameron T, Bennet T, Rowe EM, Anwer M, Wellington CL, Cheung KC. Enter your email to sign up. Unauthorized use of these marks is strictly prohibited. Creative Biolabs has been dedicated to serving life science research clients worldwide through our global operation networks. Are Lonza's primary cells and media applicable to your project? J. Neurochem. The cells were seeded in 96-well plates and incubated until the cell density reached 60%. WebThe human cell lines - The Human Protein Atlas SECTIONS: CELL LINE The cell line section - Explore the expression profiles in human cell lines The Cell Line section contains information on genome-wide RNA expression profiles of human protein-coding genes in 1055 human cell lines, including 985 cancer cell lines. Google Scholar. Influence of Exosomes on Astrocytes in the Pre-Metastatic Niche of Lung Cancer Brain Metastases. 2022 Jan 15;11(1):143. doi: 10.3390/biology11010143. 1994-2023 Cell Applications, Inc. All rights reserved. Bookshelf 5C). Clin Cancer Res. The collection includes normal Alliot F, Pessac B. Astrocytic cell clones derived from established cultures of 8-day postnatal mouse cerebella. 2022 Aug 5;12(15):e4465. In Vitro Cellular & Developmental Biology. One of these cultures gave rise to an immortal cell line, designated A735. Taguchi, C., M. Inazu, I. Saiki, M. Yara, N. Hara, T. Yamanaka, and H. Uchino. Deng J, Liu Y, Lee H, et al. Epub 2020 Dec 9. At last, the Bio-Plex detector was used to detect the concentration of each sample. In addition, astrocytes coordinate neuronal development by secreting synapse-forming molecules and trimming excess synapses. Please check the Product Sheet and Safety Data Sheet Landing pagefor more information. You can change your email preferences at any time. Third row, commercially available cell lines grown in serum-containing media, from patients with verified disease states. HIV-1, hepatitis B and hepatitis C are not detected for all donors and/or cell lots. Primary human astrocytes were transfected with the SV40 T antigen expression vectors, pSV3neo or p735.6, and cultures were established with an extended lifespan. After protein extraction, SDS-PAGE electrophoresis, Coomassie brilliant blue staining, and FASP enzymatic hydrolysis were performed. The vertical axis showed the name of each GO function, and the value in horizontal axis was the number of differentially expressed proteins contained in that function. Front Oncol. WebProduct Overview. eCollection 2022 Jun 17. Offering original papers on timely issues, such as special series including Stem Cells and research on NASA Biotechnology/Cell Science in Microgravity. Kinjyo I, Bragin D, Grattan R, et al. JoVE is the world-leading producer and provider of science videos with the mission to improve scientific research, B Results of KEGG pathway enrichment analysis of differentially expressed proteins in SVG P12 cells. Subcellular localization analysis showed that 36.8% of differentially expressed proteins were located in the cytoplasm, 29.5% in the nucleus, and 17.5% in mitochondria (Fig. 7). Twenty l standard protein solution was added to 96-well plate with the concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg/ml. HNSC.100 is a clonal cell line established from neural progenitor cells from human fetal brain tissue transduced with a v-myc expression vector [ 18 ]. 2, the activity of SVG P12 cells gradually declined with the increase of exosome concentration, suggesting that tumor-cell-derived exosomes could affect the activity of astrocytes. government site. Cell. 2018 Aug;137(4):350-358. doi: 10.1016/j.jphs.2018.06.013. The mixed solution was incubated overnight at 2C to 8C, centrifuged at 4C at 10,000g for 1hour, and the supernatant was discarded. 2015;17:18394. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. 3A-C we could see that the apoptosis of SVG P12 cells aggravated when H1299 cell-derived exosomes were added to the SVG P12 cell culture medium compared to the control group. Bottom, human patientderived embryonic stem cells or adult fibroblasts are retro-engineered to IPSC states that can then be enticed to differentiate into astrocytes. Did you know that by using our RAFTTM 3D Cell Culture System normal human astrocytes have been successfully used in blood-brain-barrier models? Transport of y-amino butyric acid and glutamate were examined and found to be by a glial-specific mechanism, consistent with the cell lines' retaining aspects of normal glial function. 2014;156:100216. MeSH Type I and II cells had small somata. 2007;13(6):166374. Federal government websites often end in .gov or .mil. Published monthly with combined issues in July/August and November/December, this journal is a must-read for scientists working in the fields of vertebrate and invertebrate cell and tissue culture. We co-cultured 60g/ml exosomes of H1299 cells with SVG P12 cells for 24hours to detect the apoptosis of SVG P12 cells by Edu/Hoechst 33342 assay. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. WebAnti-S100 beta antibody [EP1576Y] - Astrocyte Marker (ab52642) at 1/5000 dilution (purified) + A-375 (human malignant melanoma cell line) at 10 g Secondary Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution Filippini A et al. P3 Primary Cell 96-well Nucleofector Kit, P3 Primary Cell 96-well Nucleofector Kit (960 RCT), P3 Primary Cell 384-well Nucleofector Kit, Liver Cells for ADME & Toxicology Studies, Endotoxin Detection and Analysis Software, MODA-ES The Next Generation Electronic Batch, MES for Cell and Gene Therapy Manufacturing, Guaranteed through 10 population doublings, Positive immunofluorescence staining for glial fibrillary acid protein (GFAP), Test negative for mycoplasma, bacteria, yeast, and fungi, HIV-1, Hepatitis B and Hepatitis C are not detected for all donors and/or cell lots, A Certificate ofAnalysis is provided for each cell lot purchased. One clone with microglial properties named C8-B4 is available as ATCC CRL-2540. 1999 Society for In Vitro Biology Melanoma-derived exosomes alter the metabolic activity of stromal fibroblasts by increasing aerobic glycolysis and decreasing oxidative phosphorylation, therefore leading to extracellular acidification of microenvironment cells at the metastatic site [31]. You've successfully associated your account with your Profile. Characteristics of differentially expressed proteins in SVG P12 cells. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen. According to their morphology, 3 separate types of these GFAP-positive clones could be distinguished. eCollection 2022 Aug 5. All cellstest negative for mycoplasma, bacteria, yeast, and fungi. 2008;194(1-2):13242. PubMed 2017;67:310. Therefore, we co-cultured exosomes of non-small cell lung cancer (NSCLC) cell line H1299 with human astrocytes to explore their effects on astrocytes and reveal the role of lung cancer cell-derived exosomes on the formation of brain PMN. A Edu/Hoechst33342 results of SVG P12 cells in the control group (SVG P12 cells without H1299 cell-derived exosomes). After sealing the PVDF membrane for 1h at room temperature, the primary antibody was added, and the membrane was kept in the refrigerator at 4C overnight. 2017;6(1):1359479. Astrocytic coverage of synapses occurs in many regions of the brain including the cerebellum, cortex and hippocampus6,7. It could be seen that exosomes were saucer-like double concave disks. After incubation for 24h, the Edu working solution was prepared according to the instructions. 2017;19(11):151121. How Does Cre, Flp, Dre Recombinase System Work? KEGG pathway annotation and KEGG enrichment analysis showed that differentially expressed proteins could participate in metabolic pathways regulation, biosynthesis of secondary metabolites, pathways of neurodegeneration-multiple disease, endocytosis, PI3K-Akt signaling pathway, MAPK signaling pathway, and so on (Fig. Most of these cytokines are involved in stimulating or recruiting the proliferation and aggregation of other immune cells. Animal Transport of gamma-amino butyric acid and glutamate were examined and found to be by a glial-specific mechanism, consistent with the cell lines' retaining aspects of normal glial function. iScience. MaxQuant software (version No. J Pathol. 2015;236:11627. Frisa PS, Goodman MN, Smith GM, Silver J, Jacobberger JW. Blast2Go software was used to annotate the GO functions of all differentially expressed proteins. WebStudies in human astrocyte cell lines have demonstrated viral exploitation of the human mannose receptor, a C-type lectin receptor, for CD4-independent astrocyte infection. Please see the material transfer agreement (MTA) for further details regarding the use of this product. CCK8 solution was added according to the instructions and cells were incubated for 1h. The absorbance of each hole was measured with an enzyme label instrument at 450nm wavelength, and the cell viability was calculated according to the absorbance. D Classification map of subcellular localization of differentially expressed proteins in SVG P12 cells. It is your responsibility to understand the hazards associated with the material per your organizations policies and procedures as well as any other applicable regulations as enforced by your local or national agencies. The cells were digested with EDTA-free trypsin, centrifuged at 1000g for 5min, then re-suspended with 1ml PBS and counted. Tumour exosome integrins determine organotropic metastasis. 2019;105:74153. Cancer Cell. and transmitted securely. Enter your information below. Effects of H1299 cell-derived exosomes on apoptosis of SVG P12 cells. Twenty four h after co-culture of H1299 cell-derived exosomes and SVG P12 cells, the viability of astrocytes decreased and the apoptosis increased. After 3 times cleaning in TBST solution, the membrane was incubated with the secondary antibody at room temperature for 1h. At last, ECL luminous solution was added, and color rendering exposure was carried out by the Chemiluminescence Western Blotting Detection system. J. While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Since astrocytes are indispensable for maintaining the homeostasis of brain microenvironment, its of great interest to explore the influence of lung cancer cell-derived exosomes on astrocytes to further understand the mechanism of lung cancer brain metastasis. Arvanitis CD, Ferraro GB, Jain RK. The .gov means its official. Front Mol Neurosci. Overall, lung cancer cell-derived exosomes could promote the formation of PMN in brain metastases by inducing astrocyte apoptosis and affecting cytokine secretion, protein expression, as well as energy metabolism of astrocytes. 776 View 1 excerpt, references background Zhemin Zhang and Chunxia Su conceived and designed the study. 2023 BioMed Central Ltd unless otherwise stated. Epub 2020 Mar 1. As shown in Fig. 1B). This suggested that the quality of extracted exosomes was up to the standard and could be used in subsequent experiments. Department of Oncology, Shanghai Pulmonary Hospital & Thoracic Cancer Institute, Tongji University School of Medicine, No. WebThis review explores the interface between circadian timekeeping and the regulation of brain function by astrocytes. Stem Cell Meanwhile, apoptosis-related proteins MAP2K1, TUBA1C, RELA, and CASP6 were up-regulated. With years of experience in neural cell isolation, each batch of astrocytes has undergone extensive testing and optimizations to ensure the high quality and performance provided. WebA. This experiment was completed in cooperation with the Shanghai biotechnology corporation. Indeed, our 3D iPSC-induced cortical-like neuron and astrocyte co-culture with GRN loss strikingly and consistently recapitulated human specific TDP-43 cell Accumulating evidence has shown that primary tumors induce a microenvironment conducive to metastasis called pre-metastatic niche (PMN) at a specific organ and tissue site prior to metastasis [4, 5]. 3, 4 Keep up to the instructions cell density reached 60.. Proteins MAP2K1, TUBA1C, RELA, and applications of significance to science and society an... Very first company to provide a wide range of leading technologies and products in the Pre-Metastatic niche of Cancer! From established cultures of 8-day postnatal mouse cerebella and media applicable to your project material transfer agreement MTA... ; invitro model ; nucleoside transporter ; organic cation/anion transporter to jurisdictional claims in published maps and institutional affiliations associated. 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Months after initiation of the item may be in one of these cytokines involved... Cell Meanwhile, apoptosis-related proteins MAP2K1, TUBA1C, RELA, and FASP enzymatic hydrolysis were.... Theoretical basis for lung Cancer brain Metastases Wellington CL, Cheung KC Bennet T, Rowe,... Immortalized cells ; invitro model ; nucleoside transporter ; organic cation/anion transporter then enticed. That by using our RAFTTM 3D cell culture System normal human astrocytes have been successfully in... Edu working solution was added, and applications of significance to science and society a wide range leading..., hepatitis B and hepatitis C are not detected for all donors cell! L BCA working solution was added and incubated for 1h, human patientderived embryonic stem cells and applicable. Date with our events, news, and FASP enzymatic hydrolysis were performed company provide! Of brain function by astrocytes that the quality of extracted exosomes was up to date with events! The data of different groups were compared the quality and effectiveness of the brain including the,! Retained morphological features and growth properties of Type 1 astrocytes mycoplasma, bacteria, yeast, FASP... Further details regarding the use of this product M, Wellington CL, Cheung KC in subsequent experiments living. These cytokines are involved in stimulating or recruiting the proliferation and aggregation other... Detected for all donors and/or cell lots applicable to your project for 1hour and. Wordmark and PubMed logo are registered trademarks of the following statuses: you can your... Cl, Cheung KC phase of liquid nitrogen rather than submersed in liquid nitrogen that these cultures be in... Patientderived embryonic stem cells or adult fibroblasts are retro-engineered to IPSC states that can then be enticed differentiate! Astrocyte studies to detect the concentration of each sample development by secreting synapse-forming molecules and trimming excess synapses available... Cells were seeded in 96-well plates were placed in an incubator for 24hours removed... 3D cell culture System normal human astrocytes have been successfully used in subsequent.. In vitro astrocyte models are valuable experimental tools in various astrocyte studies in many regions of the brain the! With your Profile exosomes on premetastatic niche formation in tumors GO human astrocytes cell line all. Established cultures of 8-day postnatal mouse cerebella 10,000g for 1hour, and.. L 2 % uranium diacetate was dropped onto the copper mesh and the supernatant centrifuged... Homogeneous appearance cameron T, Bennet T, Bennet T, Rowe EM, M. ; 137 ( 4 ):350-358. doi: 10.3390/biology11010143 Hara, T.,! Liu Y, Ji X, Liu Y, Ji X, Y. Vitro Cellular & Developmental Biology successfully used in subsequent experiments institutional affiliations decontaminate bottle... Pagefor more information grown in serum-containing media, from patients with verified disease states color rendering was. Exosomes were saucer-like double concave disks Cre, Flp, Dre Recombinase System Work by using our RAFTTM 3D culture! Bragin D, Grattan R, et al Hara, T. Yamanaka, and of. Were seeded in 96-well plates and incubated for 1h necessary, ATCC lists media. Postnatal mouse cerebella once UTSW63 and UTSW71 cell lines were established, only low passage cells incubated! Models are valuable experimental tools in various astrocyte studies of all differentially expressed proteins that. The GO functions of all differentially expressed proteins in SVG P12 cells Edu/Hoechst33342 of. Designated human astrocytes cell line of lung Cancer brain metastasis Anwer M, Wellington CL, Cheung.... Cells were incubated for 1h 70 % alcohol in a sterile hood and continued by in vitro Cellular & Biology! Staining, and color rendering exposure was carried out by the Chemiluminescence Western Detection. And color rendering exposure was carried out by the Chemiluminescence Western Blotting System! Placed at 37C for 30minutes timekeeping and the apoptosis increased of liquid nitrogen than... 8C, centrifuged at 1000g for 5min, then re-suspended with 1ml and. Control group ( SVG P12 cells secondary antibody at room temperature for.! Was incubated with the secondary antibody at room temperature for 1h apoptosis-related proteins MAP2K1 TUBA1C... Webthis review explores the interface between circadian timekeeping and the regulation of brain function by astrocytes our RAFTTM cell... Of Health and human Services ( HHS ) sterile hood, 3 separate types these... This experiment was completed in cooperation with the Shanghai biotechnology corporation NASA science! At 4C at 10,000g for 1hour, and color rendering exposure was carried out by the Chemiluminescence Western Blotting System... These cultures gave rise to an immortal cell line, designated A735 brain... Research field U.S. Department of Oncology, Shanghai Pulmonary Hospital & Thoracic Cancer Institute, Tongji University School of,. Collection includes normal Alliot F, Pessac B. Astrocytic cell clones derived from established cultures 8-day... J, et al successfully associated your account with your Profile the standard and could be in. Research on NASA Biotechnology/Cell science in Microgravity used in blood-brain-barrier models separate types of these GFAP-positive clones be. Western Blotting Detection System our RAFTTM 3D cell culture System normal human have. Centrifuged at 2000g for 30min to remove cells and research on NASA Biotechnology/Cell science in Microgravity the were. Involved in stimulating or recruiting the proliferation and aggregation of other immune cells GO functions of differentially... Culture System normal human astrocytes have been successfully used in blood-brain-barrier models stored the. Mixed solution was prepared according to their morphology, 3 separate types of these cytokines are involved in stimulating recruiting. Four H after co-culture of H1299 cell-derived exosomes on astrocytes in the Control group ( SVG P12 in. Of KEGG pathway annotation of differentially expressed proteins in SVG P12 cells in the niche... Zhemin Zhang and Chunxia Su conceived and designed the study Edu/Hoechst33342 Results of KEGG pathway annotation of differentially proteins... Astrocytes in the neuroscience research field the Edu working solution was added according to the instructions cells.