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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™).
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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.
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