* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Isolation of zebrafish neurons using the
Survey
Document related concepts
Cortical cooling wikipedia , lookup
Stimulus (physiology) wikipedia , lookup
Subventricular zone wikipedia , lookup
Feature detection (nervous system) wikipedia , lookup
Single-unit recording wikipedia , lookup
Neurophilosophy wikipedia , lookup
Multielectrode array wikipedia , lookup
Metastability in the brain wikipedia , lookup
Optogenetics wikipedia , lookup
Neural engineering wikipedia , lookup
Nervous system network models wikipedia , lookup
Clinical neurochemistry wikipedia , lookup
Electrophysiology wikipedia , lookup
Neuroanatomy wikipedia , lookup
Channelrhodopsin wikipedia , lookup
Development of the nervous system wikipedia , lookup
Transcript
#9530 DRAFT Sharing ideas, sharing success For users, by users Customer protocol Isolation of zebrafish neurons using the gentleMACS™ Dissociator Dawn M. Johnson and Joshua A. Maurer* Department of Chemistry, Washington University in St. Louis, Missouri, USA * Corresponding author ([email protected]) • H ank`s Balanced Salt Solution without Ca2+ and Mg2+ (HBSS (w/o)) • Hank`s Balanced Salt Solution with Ca2+ and Mg2+ (HBSS (w)) Methods Sample preparation 1. Bleach zebrafish embryos immediately after collection at day 0 in 0.5% bleach solution for 2 minutes. 2. Rinse well with embryo medium and store in a 28.5 °C incubator until dissociation. 3. Treat embryos in embryo medium containing pronase (2 mg/mL) to aid removal of the chorion during dissociation by swirling for approximately 1 minute. 4. Rinse embryos several times with embryo medium. Proceed with dissociation. Background Zebrafish have long been a model system for the study of vertebrate development. However, the isolation of embryonic zebrafish neurons for in vitro studies is often a difficult and tedious process due to the inconsistencies associated with manual dissociation methods. With the incorporation of the gentleMACS™ Dissociator system, we are able to easily generate reproducible single-cell suspensions of zebrafish neurons for use in studies of neuronal guidance cues. These studies should allow for the correlation of in vitro biochemical data with the extensive collection of data on zebrafish development. Dissociation 1. Prepare enzyme mix 1 (50 μL Solution 1 and 1900 μL Solution 2 from the Neural Tissue Dissociation Kit (P)) and warm to 37°C. 2. Place the pre-bleached and pronase-treated embryos (approximately 100 embryos per C Tube) in 3 mL HBSS (w/o) with as little embryo medium as possible. 3. Transfer the embryos, with as little HBSS (w/o) as possible, to a gentleMACS C Tube containing 1950 μL pre-warmed enzyme mix 1. 4. Tightly close C Tube and attach upside down onto the sleeve of the gentleMACS Dissociator. Run the gentleMACS Program m_brain_01. 5. Incubate sample for 15 minutes at 37 °C under slow, continuous rotation using the MACSmix Tube Rotator. 6. Attach C Tube onto the sleeve of the gentleMACS Dissociator and run program m_brain_02. 7. Prepare enzyme mix 2 (20 μL Solution 3 and 10 μL Solution 4 from the Neural Tissue Dissociation Kit (P)) and transfer it into the C Tube. Invert gently to mix. 8. Incubate sample for 10 minutes at 37 °C under slow, continuous rotation using the MACSmix Tube Rotator. Materials and methods Materials • gentleMACS Dissociator or gentleMACS Octo Dissociator • gentleMACS C Tubes • MACSmix™ Tube Rotator • Neural Tissue Dissociation Kit (P) • Dead Cell Removal Kit • MS Column • Pre-Separation Filters, 30 µm • Incubator • 0.5% bleach solution • Embryo medium • Pronase • Neurobasal® media (50 mL supplemented with 500 µL penicillin/streptomycin (10,000 units/mL penicillin G sodium and 10,000 µg/mL streptomycin sulfate in 0.85% saline), 1 mL B27 supplement, and 500 µL GlutaMAX™). 1/2 The content of this publication has not been verified by Miltenyi Biotec. 9. A ttach C Tube onto the sleeve of the gentleMACS Dissociator and run program m_brain_03 followed by m_brain_01. 10.Incubate sample for 10 minutes at 37 °C under slow, continuous rotation using the MACSmix Tube Rotator. 11. Resuspend sample and apply the cell suspension to a Pre-Separation Filter, 30 µm, placed on a 15 mL tube. 12.Wash the C Tube with 10 mL HBSS (w) and apply to the Pre-Separation Filter. 13.Discard the Pre-Separation Filter and centrifuge cell suspension at 300×g for 10 minutes at room temperature. Aspirate supernatant completely. 14.Proceed with the Dead Cell Removal Kit. 25 µm Use of the Dead Cell Removal Kit 1. Resuspend cell pellet in 100 μL Dead Cell Removal MicroBeads. 2. Mix well and incubate for 15 minutes at room temperature. 3. Place an MS Column in the magnetic field of a suitable MACS® Separator. 4. Rinse column with 500 μL of 1× Binding Buffer. 5. Add 500 μL of 1× Binding Buffer to the cell suspension. 6. Apply cell suspension to the column. Let the live cells pass through. 7. Rinse the column with 4× 500 μL of 1× Binding Buffer. Collect effluent as live cell fraction. 8. Centrifuge at 300×g for 10 minutes. 9. Aspirate supernatant completely and resuspend in 1 mL fully supplemented Neurobasal media. 25 µm Figure 1: Phase-contrast images of zebrafish neurons imaged live at day 1 (above) and fixed at day 4 (below). Results Conclusion Through the use of the gentleMACS Dissociator system in conjunction with the Neural Tissue Dissociation Kit (P) and the Dead Cell Removal Kit, we were able to achieve a single‑cell suspension of zebrafish neurons. Figure 1 shows live cell image 24 hours after plating in fully supplemented Neurobasal media and fixed neurons after 4 days in culture. Isolation of zebrafish neurons can be accomplished with ease using the gentleMACS Dissociator. Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec contact. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. gentleMACS, MACS, and MACSmix are either registered trademarks or trademarks of Miltenyi Biotec GmbH. All other trademarks mentioned in this document are the property of their respective owners and are used for identification only. Copyright © 2012 Miltenyi Biotec GmbH. All rights reserved. 2/2 The content of this publication has not been verified by Miltenyi Biotec. V. 01 Visit www.gentleMACS.com for more information on Miltenyi Biotec's sample preparation portfolio or find more customer protocols on www.gentleMACS.com/protocols