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Magnetic-activated cell sorting (MACS)
can be used as a large-scale method for
establishing zebrafish neuronal cell
cultures
Georg Welzel
1,2
, Daniel Seitz
1,2
& Stefan Schuster
1,2
1
Department of Animal Physiology, University of Bayreuth, 95440 Bayreuth, Germany,
2
Friedrich-Baur BioMed Center, 95448
Bayreuth.
Neuronal cell cultures offer a crucial tool to mechanistically analyse regeneration in the nervous system.
Despite the increasing importance of zebrafish (
Danio rerio
)asan
in vivo
model in neurobiological and
biomedical research,
in vitro
approaches to the nervous system are lagging far behind and no method is
currently available for establishing enriched neuronal cell cultures. Here we show that magnetic-activated
cell sorting (MACS) can be used for the large-scale generation of neuronal-restricted progenitor (NRP)
cultures from embryonic zebrafish. Our findings provide a simple and semi-automated method that is likely
to boost the use of neuronal cell cultures as a tool for the mechanistic dissection of key processes in neuronal
regeneration and development.
Neural repair and regeneration in the central nervous system (CNS) is a promising topic in regenerative
medicine, with targets ranging from the treatment of spinal cord injuries to that of stroke and degen-
erative brain diseases such as Alzheimer and Parkinson’s
1
. Significant progress in developing new
therapeutic strategies might be achieved by studying the zebrafish, a vertebrate whose CNS has a much higher
regenerative capacity than that of humans or of other mammals
2
. The regulation and maintenance of adult
neurogenic regions in the brain of this fish and its ability to even completely regenerate injured brain regions
already make the zebrafish an outstanding in vivo model to study the processes of neural development, adult
neurogenesis and neural regeneration in vertebrates
2,3,4,5
.
In order to further dissect molecular mechanisms involved in the regenerative capacities, working on neuronal
cell cultures would be a powerful additional tool. However, despite the enormous success of zebrafish as an in vivo
model system, only a few attempts have been reported so far describing the effective culture of primary neuronal
cells from embryonic to adult zebrafish
6,7,8,9,10
. Moreover, the challenging and time-consuming methods currently
used for manual dissection of embryonic neural tissues only permit the processing of a limited number of
embryos. Furthermore, these do not yet allow the robust establishment of standardised neuronal cultures but
rather result in mixed cell cultures
6,7,8,9
even when amended with fluorescence-activated cell sorting
8
. In mam-
mals, enriched neuronal cell cultures can be reliably generated by using magnetic-activated cell sorting (MACS).
Since the polysialilated form of the neural cell adhesion molecule (PSA-NCAM) is a distinct marker of immature
neuronal-restricted progenitors (NRPs)
11–13
, MACS with microbeads conjugated to an antibody against PSA-
NCAM can be used to generate cultures of mammalian NRPs
14,15
, which subsequently differentiate into neurons
but not glial cells
11–13
.
Here we show for the first time the successful application of a MACS based technique in zebrafish. By using a
semi-automated dissociation process along with anti-PSA-NCAM microbeads, we isolated immature neuronal
cells from a large number of embryonic zebrafish. Our simple, cheap and reproducible method allows the large-
scale generation of enriched and viable in vitro cultures of zebrafish NRPs and lays the ground for the establish-
ment of differentiated neuronal cell cultures that will be useful to study neurogenesis or axonal regeneration.
Results
Primary cell cultures derived from zebrafish embryos contain few neural cells.To establish neuronal cell
cultures from zebrafish, we first dissociated sterilized zebrafish embryos at 30 hours post fertilization (hpf) into a
OPEN
SUBJECT AREAS:
BIOLOGICAL
TECHNIQUES
NEUROLOGICAL MODELS
EXPERIMENTAL ORGANISMS
Received
19 September 2014
Accepted
29 December 2014
Published
22 January 2015
Correspondence and
requests for materials
should be addressed to
G.W. (georg.welzel@
uni-bayreuth.de) or
S.S. (stefan.schuster@
uni-bayreuth.de)
SCIENTIFIC REPORTS | 5 : 7959 | DOI: 10.1038/srep07959 1
single cell suspension by applying a semi-automated and standar-
dised protocol (see Methods). We then cultured the cells on laminin
in a defined serum-free medium especially formulated for neural cell
cultivation. Since we used entire zebrafish embryos, the cultivation of
the dissociated cells resulted in heterogeneous cell cultures with
various cell morphologies (Fig. 1a). As in blastula-derived cell
cultures
6
, the embryonic cells also started to form interconnected
cell aggregates after a few days in vitro (Fig. 1a). The embryonic
cells were cultured in a medium that promotes growth and survival
of neural cells. Additionally, laminin was employed as a substrate
that enhances neural differentiation and survival
16
. Nevertheless,
only a small proportion of cells could be identified as neuronal
after one week of culture and only single cells both within and
beyond the aggregates expressed neuronal and glial markers
(Fig. 1b, c).
Isolation of PSA-NCAM positive cells from embryonic zebrafish
by using MACS.As illustrated by Fig. 1, a method is needed to
specifically enrich neuronal cells in zebrafish embryonic cell
populations. To separate neuronal cells from the heterogeneous
single cell suspension, we attempted to use magnetic-activated cell
sorting (Fig. 2a) with anti-PSA-NCAM microbeads, a cost-efficient
technique that is widely applied in mammals
14,15
. Because the
antibody used in this magnetic-based isolation targets polysialic
acid (PSA) and because PSA-NCAM is expressed in both
embryonic
17,18
and adult zebrafish CNS
19,20
we were hoping that
anti-PSA-NCAM microbeads could be applicable in zebrafish as
well. To test this assertion, we used the same PSA-NCAM
antibody that would later be conjugated to the microbeads to
detect PSA-NCAM immunoreactivity on paraffin sections from
embryonic zebrafish at 30 hpf. The findings clearly demonstrated
the expression of PSA-NCAM in cells of the developing zebrafish
CNS (Fig. 2b,c), raising hope that anti-PSA-NCAM microbeads
could be used to sort neuronal cells in zebrafish.
When we thus performed a magnetic-based separation, we
obtained a positive cell fraction with magnetically labelled cells and
an unlabelled negative cell fraction (Fig. 2a). We cultured cells from
each fraction (original, negative, positive) on laminin in a defined
serum-free medium for further in vitro characterization. Since the
isolated cells were exposed to mechanical and physical stress during
dissociation and magnetic separation, it was crucial to test first the
viability of the cells in the positive fraction. Light microscopic exam-
ination revealed that the initially round shaped cells of the positive
fraction became adherent and extended long neurites resulting in
unipolar, bipolar or multipolar morphologies similar to NRPs in
mammalian cell cultures
11
. The cells were migratory and started to
reorganize in small clusters and to form discrete cell aggregates that
were interconnected by bundles of neurites after 24 to 48 h of culture
(Fig. 2d). Furthermore, the apparently homogeneous neuronal cul-
tures formed from the positive fraction showed high viability and
metabolic activity (Fig. 2e–g). In contrast, primary cultures from the
original and the negative fraction appeared more heterogeneous and
showed lower metabolic activity (Fig. 2g).
Generation of highly enriched NRP cultures.The findings thus
suggest that our magnetic microbead-based isolation technique
Figure 1
|
Without further treatment zebrafish embryonic cell cultures contain only few neuronal cells. (a) Embryonic zebrafish at 30 hours post
fertilization (hpf) were dissociated into a single cell suspension by a semi-automated dissociation process (see Methods). The primary cells were cultured
on laminin (50.000 cells/cm
2
) in a defined serum-free MACS Neuro Medium. Representative light microscope images of the in vitro cultures show cells
with different morphologies, which started to form cell aggregates that became interconnected by neurites after a few days in vitro. (b) Immunofluorescent
staining of 48 h and (c) 7 d primary cultures identified few neuronal (neurofilament, NF) and glial (glial fibrillary acid protein, GFAP) cell types located
within these aggregates. Nuclei were stained with DAPI (blue). All scale bars, 50 mm.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 5 : 7959 | DOI: 10.1038/srep07959 2
indeed generated enriched and homogeneous NRP cultures. To
further test this, we identified NRPs in the positive fraction and
quantified the purity of the enriched cultures by monitoring
the expression of PSA-NCAM (Fig. 3a) as well as of non-
phosphorylated neurofilaments (NF; Fig. 3b), a pan-neuronal
intermediate filament found specifically in adult and developing
neurons, including neuronal progenitor cells. These assays proved
that magnetic separation with anti-PSA-NCAM microbeads indeed
led to a significant enrichment of PSA-NCAM positive cells in the
positive fraction (70.9 67.1% after 3 h and 77.0 69.1% after 15 h,
respectively; P,0.001) compared to the original fraction (3.8 6
1.3% after 3 h and 3.2 62.1% after 15 h, respectively). As a control
the negative fraction showed the depletion of PSA-NCAM
1
cells (0.4
60.3% after 3 h and 0.9 60.6% after 15 h, respectively; Fig. 3e).
Beside the expression of PSA-NCAM, we were also able to detect the
expression of NFs (Fig. 3b). We demonstrated the significant
enrichment of NF positive cells in the positive fraction (72.3 6
5.2%; P,0.001) compared to the original fraction (5.4 61.0%).
As a control, the negative fraction showed a reduction of neuronal
cells (1.1 60.7%; Fig. 3e).
We subsequently characterized the remaining 27% of NF-negative
cells. Since we used a culture medium that promotes the survival of
neural cells, we hypothesized that the majority of the non-neuronal cells
were glial cells. We determined the number of glial cells in the positive
fraction by their expression of glial fibrillary acid protein (GFAP; Fig. 3c),
an intermediate filament that is expressed in mature astrocytes as well as
in neural or glial progenitor cells
21
. As PSA-NCAM can also be expressed
in oligodendrocyte progenitors
15,22
, we identified these and other glial
progenitors by using an antibody against a ganglioside-specific antigen
(A2B5; Fig. 3d), a marker for glial-restricted progenitors (GRPs)
23
that
has already been described in the brain of other teleost fish
24,25
.The
staining revealed that most of the NF negative cells expressed GFAP
(17.5 64.0%) and A2B5 (11. 9 62.7%) (Fig. 3e). Taken together, the
glial assays suggest that the remaining, non-NF positive cells in our
cultures were predominantly PSA-NCAM positive GRPs.
Overall, the immunocytochemical analysis of the MACS-isolated
positive cell population showed that our novel magnetic cell sorting
method worked, allowing us to successfully generate viable, enriched
NRP cultures from embryonic zebrafish.
Enriched NRP cultures form neuronal aggregates in vitro.As our
objective was to generate long-term neuronal cell cultures from
zebrafish, we next aimed to characterize the cells in our NRP cultures
beyond 3 h in vitro. Therefore, cells from the neuronal enriched positive
fraction were cultured for 2 and 7 d, respectively. As already shown, the
cells in the positive fraction started to form aggregates that became
interconnected by neurites within 2d(Fig.2d).Duringthefollowing
days, both the size of the aggregates and the density of neurites
extending from the aggregates increased (Fig. 4a). Since we started
with cell cultures containing predominantly NRPs (Fig 3a, d), we
Figure 2
|
Homogeneous and viable cell cultures of MACS-isolated PSA-NCAM positive cells from embryonic zebrafish. (a) Schematic representation
of magnetic-activated cells sorting (MACS) via magnetic anti-PSA-NCAM microbeads. The single cell suspension of dissociated embryonic
zebrafish was incubated with anti-PSA-NCAM microbeads and loaded onto a MACS Column. The magnetically labelled cells were retained in the
magnetic field of a MACS Separator, whereas the unlabelled cells flowed through the column (negative fraction). The labelled PSA-NCAM positive cells
were then flushed out and collected (positive fraction). (b) Coronal and (c) transversal paraffin sections of embryonic zebrafish (30 hpf) showing PSA-
NCAM expression (green) in the tail (grey arrow) as well as in several regions of the brain (white arrows) and the eyes (red arrow). Note the
autofluorescence of the yolk. Nuclei were stained with DAPI (blue). tel, telencephalon; di, diencephalon. (d) Primary cultures of adherent cells from the
positive fraction (50.000 cells/cm
2
) showed unipolar (white arrow), bipolar (black arrow) and multipolar (red arrow) morphologies and formed
interconnected cell aggregates after 24 h. (e) Viability was assessed by using the Live/Dead assay showing live cells stained with calcein (green) and dead
cells with EthD-1 (red). All scale bars, 25 mm. (f) Cells from each fraction showed high viability at 3 h in vitro (n53, mean 6SD). (g) Metabolic activity of
cells from each fraction was detected by a resazurin-based assay at 3 h, 24 h and 48 h in vitro (n55, mean 6SD, P ,0.001). See Fig. 3 for further
characterization of PSA-NCAM positive cells and their viability.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 5 : 7959 | DOI: 10.1038/srep07959 3
expected that the majority of the cells forming these aggregates are still
neuronal after 7 d in vitro. To confirm this assumption, we investigated
the cellular composition of the aggregates by immunocytochemistry.
Consistent with our findings at 3 h (Fig. 3), the cells showed positive
immunostaining for PSA-NCAM and NF (Fig. 4b, c), whereas GFAP
staining was evident in only a few cells (Fig. 4d).
The predominant expression of PSA-NCAM along with NF vali-
dates the neuronal composition of the aggregates after 2 as well as 7 d
in vitro (Fig. 4c, d). Overall, zebrafish NRPs can be enriched by
MACS and form in vitro neuronal aggregates, in contrast to hetero-
geneous cell aggregates developed without MACS.
Neuronal network formation induced by retinoic acid.In mouse
26
,
rat
11,13
or human
27
homogenous cell populations of neuronal-restricted
progenitors can be used to develop cell cultures of differentiated and
functional neurons. Here, retinoic acid (RA) induces the in vitro
differentiation into multiple neuronal cell types. To test whether our
NRPs isolated from zebrafish embryos would similarly respond to RA,
we grew enriched NRP cultures in a differentiation medium containing
1mM RA. After several days under these conditions, several cells began
to leave the aggregates, forming a monolayer and displaying neuronal
morphologies with small extending processes (Fig. 5a). As expected,
the immunocytochemical analysis of these cells revealed the expression
of NFs (Fig. 5b). During the next weeks, further neuronal maturation
and the formation of neuronal networks of NF expressing cells were
observed (Fig. 5c, d). In addition, NF positive cells seemed to be
connected to adjacent neurons indicating the formation of synapses
(Fig. 5d). Therefore, the development of neuronal networks provides
Figure 3
|
Enrichment of immature neuronal-restricted progenitors using PSA-NCAM mediated magnetic cell separation. Cells from the original, the
negative and the positive fraction were cultured on laminin (50.000 cells/cm
2
). After 3 h in vitro, the expression of (a) polysialilated-neural cell
adhesion molecules (PSA-NCAM), (b) neurofilaments (NF), (c) glial fibrillary acid proteins (GFAP) and (d) A2B5 was analysed immunocytochemically
in each cell fraction. Nuclei were stained with DAPI (blue). Scale bar, 25 mm. (e) The positive fraction showed a significantly higher content of PSA-
NCAM- and NF-positive cells (n 55, mean 6SD, P ,0.001). GFAP was detected in glial cells in all fractions (n55, mean 6SD, P50.41). A2B5 on the
surface of glial-restricted progenitors (GRPs) is only detected in the positive fraction (n55, mean 6SD, P50.001).
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 5 : 7959 | DOI: 10.1038/srep07959 4
evidence suggesting that RA promotes the differentiation of NRPs into
mature neurons in zebrafish as well.
Taken together, our findings suggest that our method has pro-
duced viable neuronal cells that not only respond appropriately to
RA differentiation cues but form typical morphologies and
interconnections.
Discussion
The currently available methods for establishing neuronal cell cul-
tures in zebrafish are insufficient for large-scale experiments. The
labour-intensive and time-consuming microdissection of neural tis-
sues only allow the processing of a small number of embryos and the
resulting cultures contain only a small fraction of neuronal cells
6,7,8,9
.
We have employed a semi-automated dissociation that allows the
simultaneous processing of a large number of zebrafish embryos in
less than an hour. Nevertheless, culturing the resulting cell popu-
lation led to a heterogeneous primary cell culture, similarly as in
blastula-derived cell cultures
6
. We therefore used magnetic-activated
cell sorting (MACS) to specifically enrich neuronal cells in zebrafish
cell populations. We show that magnetic anti-PSA-NCAM microbe-
ads are applicable for zebrafish and can easily be used to separate
neuronal-restricted progenitors (NRPs) for subsequent long-term
neuronal cell cultures. Because MACS is comparatively simple and
cost-effective to establish, compared e.g. to fluorescent-activated cell
sorting (FACS), our methods would be suitable for many labs. If a
higher purity is required, our findings suggest that a first major step
should be the depletion of PSA-NCAM expressing glial-restricted pro-
genitors (GRPs) by using MACS along with anti-A2B5 microbeads.
In summary, we have established a novel easily applicable large-
scale method that many labs could use to generate enriched and
viable standardised neuronal cell cultures from embryonic zebrafish
in only 2 hours. Large-scale in vivo screens of chemical libraries are a
powerful tool in studies on neural degeneration, regeneration and
neural development in zebrafish
28,29
. However, many of this studies,
like the search for intrinsic and extrinsic factors that underlie the
ability of zebrafish neurons to functionally regenerate axons
30
or
reestablish neuronal circuits and behavior
31
, are likely to benefit from
an in vitro system that allows drug screening experiments. In general,
our technique should be particularly useful for in vitro studies to
address the mechanistic basis of the high neuroregenerative potential
in zebrafish. Particularly, we expect that findings from zebrafish
NRPs will facilitate the improvement of therapeutic strategies, such
as cell-replacement therapies, where cultured NRPs could be a prom-
ising exogenous source of replacement neurons
12,27,32
.
Methods
Fish strain and maintenance.Zebrafish (Danio rerio) wild-type Tuebingen (Tu)
strain were maintained at 28.5uC under standard conditions
33
. Fertilized eggs were
collected and washed with 0.5% bleach solution for 1 min. Until dissociation, the
embryos were kept and raised in E3 embryo medium, as described
33
. The embryonic
development was staged by hours post fertilization (hpf) according to Kimmel et al.
34
.
Since the expression of PSA-NCAM is highest from 27–40 hpf
17
, neuronal-restricted
progenitors were isolated from prim-15 (30 hpf) stage embryos.
Isolation and cell culture of neuronal-restricted progenitors (NRPs).The embryos
were sterilized prior to dissociation with a 70% ethanol solution for 5 sec and rinsed
three times in sterile E3 embryo medium. All subsequent steps were performed under
sterile conditions. First, the embryos were pretreated with a dilute solution of pronase
E (2 mg/ml in E3 embryo medium; Sigma) for 1 min to facilitate the dechorionation
during dissociation and washed again three times. The embryos were then dissociated
automatically into a single cell suspension with the MACS Neural Tissue Dissociation
Kit (T) (Miltenyi Biotec) in gentleMACS C tubes on the gentleMACS Dissociator
(Miltenyi Biotec) according to the manufacturer’s recommended protocol. Briefly, up
to 1000 sterilized and pronase-treated embryos were transferred into one
Figure 4
|
Spontaneously formed aggregates predominantly contain neuronal cells. (a) NRP cultures from embryonic zebrafish were cultured for 2 and
7 d, respectively. Representative images show the formation of cell aggregates that became interconnected by neurites. Both the size of the aggregates and
the density of neurites extending from the aggregates increased until day 7. Immunocytochemical analyses demonstrated the expression of (b)
polysialilated-neural cell adhesion molecule (PSA-NCAM) and (c) neurofilaments (NF) in most of the cells within the aggregates at 2 and 7 d in vitro. (d)
Only a few cells showed the expression of glial fibrillary acid proteins (GFAP). Nuclei were stained with DAPI (blue). All scale bars, 50 mm.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 5 : 7959 | DOI: 10.1038/srep07959 5
gentleMACS C-Tube (Miltenyi Biotec) containing Hanks’ balanced salt solution
without calcium or magnesium (HBSS w/o; PAA Laboratories). HBSS w/o was
aspirated completely and the dissociation was started by adding 1950 ml of enzyme
mix 1 and by using the defined gentleMACS program m_brain_01 on the
gentleMACS Dissociator. The sample was incubated for 15 min at 37uC followed by
the gentleMACS program m_brain_02. After adding 30 ml of enzyme mix 2, the
sample was incubated for 10 min at 37uC followed by the gentleMACS program
m_brain_03. Subsequent to an additional incubation for 10 min at 37uC, the sample
was applied to a cell strainer placed on a 50 ml tube. The cell strainer was washed with
10 ml of HBSS and the resulting single cell suspension was centrifuged at 300 3gfor
10 min.
PSA-NCAM
1
NRPs were then purified with magnetic-activated cell sorting
(MACS) and MACS anti-PSA-NCAM Microbeads (Miltenyi Biotec) according to the
manufacturer’s recommended protocol. Briefly, 10
7
cells were resuspended in 70 mlof
cold separation buffer (phosphate-buffered saline (PBS) containing 0.5% bovine
serum albumin (BSA) and 2 mM EDTA; both from Sigma-Aldrich), incubated for
15 min at 4uC with 20 ml of anti-PSA-NCAM Microbeads per 70 ml of cell suspen-
sion, washed by adding 2 ml of separation buffer and centrifuged at 300 3g for
10 min. Cells were resuspended in 500 ml of separation buffer and applied onto a
MACS Column (MS type; Miltenyi Biotec) placed in the magnetic field of a MACS
Separator (Miltenyi Biotec). The flow-through was collected as the unlabelled nega-
tive fraction. The column was then washed three times with 500 ml of separation
buffer and the retained magnetically labelled cells were flushed out with 1 ml of
separation buffer as the positive fraction (Fig 2a). Cells from the original, the negative
and the positive fraction were counted using a particle size counter (Z2, Beckman
Coulter GmbH) and seeded onto laminin-coated (50 mg/ml; Sigma) plastic plates or
sterilized glass slides for immunocytochemistry (50.000 cells/cm
2
). Cells from each
fraction were cultured in serum-free MACS Neuro Medium (Miltenyi Biotec) sup-
plemented with 1 3MACS NeuroBrew-21 (Miltenyi Biotec), 1% GlutaMAX and 1%
penicillin–streptomycin (Gibco). Cultures were incubated at 28.5uC and 5% CO
2
in a
humidified environment.
To induce neuronal differentiation, retinoic acid (1 mM in DMSO; Sigma) was
added to the medium 1 day after seeding. Half of the medium was changed every two
to three days.
Immunocytochemistry.All staining procedures were carried out at room
temperature. Cultures from the original, the negative and the positive cell fraction
were chemically fixed for 10 min using a 4% formaldehyde solution in PBS and
washed three times with PBS. In order to detect cytoskeletal components, cells were
permeabilized with 0.1% Triton-X 100 in PBS for 5 min. Unspecific binding was
blocked with PBS containing 5% normal goat serum (NGS; Sigma-Aldrich) for 1 h.
Samples were incubated with a mouse monoclonal antibody against polysialilated
neuronal cell adhesion molecule (PSA-NCAM, 1:100; Miltenyi Biotec), non-
phosphorylated neurofilaments (NF, clone SMI-311, 1:1000; abcam), glial fibrillary
acid protein (GFAP, clone ZRF-1, 1:1000; abcam) or A2B5 (clone 105, 1:500; Sigma)
for 1 h. After washing three times with PBS, cells were incubated with an Alexa Fluor
488-conjugated goat anti-mouse secondary antibody (1:400, Invitrogen) for 1 h.
Slides were washed three times in PBS and mounted using ProLong Gold antifade
reagent with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen).
Immunocytochemically and immunohistochemically stained slides were visualized
using a fluorescent microscope (Axioplan 2 imaging, Zeiss). Representative images
were recorded digitally using a ProgRes C14 camera system (ProgRes C14, Jenoptik
AG) in combination with a capture and image analysis software (AnalySIS Version
3.2). To estimate the purity of the positive fractions the percentage of PSA-NCAM
and NF positive stained cells were calculated for each cell fraction. Cells were coun ted
in five randomly chosen microscope fields with at least 150 cells taken from five
independent experiments and averaged. The percentage of stained cells was defined
as the number of cells positive for PSA-NCAM, NF, GFAP or A2B5 divided by the
total number of cells. Cell counting was performed using ImageJ software (Version
1.47, Rasband).
Immunohistochemistry.For immunohistochemical detection of PSA-NCAM in
paraffin embedded sections, zebrafish embryos (30 hpf) were first dechorionated
using two fine forceps and fixed overnight at 4uC using a 4% formaldehyde solution in
PBS. The embryos were orientated in a same direction and embedded in 1% agarose to
fix their position. Both dehydration through a series of graded ethanol and
embedding in paraffin was done automatically by a tissue processing center (TPC 15
Duo, Medite). The paraffin block was cut on a rotary microtome (Biocut 2030,
Reichert-Jung) into 5 mm sections, which were mounted on glass slides covered with
Figure 5
|
Formation of neuronal networks induced by retinoic acid (RA). (a) NRPs were cultured in a differentiation medium containing retinoic acid
(1 mM in DMSO). After 7 d in vitro many cells left the neuronal aggregates, formed adherent monolayers and extended small processes. (b) The
immunocytochemical staining of cells forming these monolayers revealed the expression of neurofilaments (NF). Nuclei were stained with DAPI (blue).
(c) After two weeks in vitro further neuronal maturation and the formation of neuronal networks were observed. (d) The NF positive neurons seemed to
be connected to adjacent neurons indicating the formation of synapses (white arrows). Nuclei were stained with DAPI (blue). All scale bars, 50 mm.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 5 : 7959 | DOI: 10.1038/srep07959 6
poly-L-lysine (Thermo Scientific). Paraffin sections were deparaffined in Roticlear
(Carl Roth GmbH) and rehydrated in a graded ethanol series (100%, 100%, 100%,
80%, 75%, 50% and distilled water for 5 min each). For heat-induced epitope retrieval
sections were incubated for 20 min with HIER H buffer (Thermo Scientific) at 95uC
in a steamer. The subsequent immunofluorescent staining protocol to detect PSA-
NCAM expression was performed as described above.
Cell viability.Viability of isolated primary cells was assessed after 3 h using a Live/
Dead two-colour fluorescence assay according to the manufacturer’s protocol
(Molecular Probes). Cells from each fraction were grown on laminin coated glass
slides. After washing with PBS, cells were exposed to 2 mM ethidium homodimer-1
(EthD-1) and 1 mM calcein-AM in PBS for 30 min at 28.5uC in the incubator. To
quantify the viability, the percentage of live cells stained with calcein (green) and dead
cells stained with EthD-1 (red) were calculated for each cell fraction. Cells were
counted in five randomly chosen microscope fields with at least 200 cells taken from
three independent experiments and averaged. The percentage of viable cells was
defined as the number of calcein-stained cells divided by the total number of cells. Cell
counting was performed using ImageJ software (Version 1.47, Rasband). Dye uptake
was detected by using a fluorescent microscope (Axioplan 2 imaging, Zeiss).
Viability of cultured cells was also determined by using a metabolic resazurin assay
(Sigma-Aldrich). Cells of each fraction were cultured in 96 well plates (15.000 cells/
well). Resazurin dye solution (4 ml) was added to each well at 3 h, 24 h and 48 h in
vitro. After 3 h of incubation at 28.5uC, the bioreduction of resazurin into red-
fluorescent resorufin by living cells was measured at each time point by fluorescence
intensity at 590 nm (excitation 560 nm) on a microplate reader (PolarSTAR
OPTIMA, BMG-Labtech). Subsequent to the resazurin assay, the total DNA content
was quantified in each well using the Quant-iT
TM
PicoGreenHdsDNA assay kit
(Invitrogen) in accordance with the manufacturer’s protocol.
Data analysis and statistics.All data was normally distributed (Shapiro–Wilk,
P.0.05) and presented as mean values 6standard deviations (SD). The statistical
significance between groups of data was evaluated by one-way ANOVAs followed by
Tukey post hoc tests. Values of P,0.05 were considered statistically significant.
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Acknowledgments
This work is supported by a Reinhart-Koselleck project of the Deutsche
Forschungsgemeinschaft (S.S.) and by grants from the Friedrich-Baur Foundation (S.S.).
We thank Antje Halwas (Schuster lab) for excellent technical help.
Author contributions
G.W. performed and analysed experiments, D.S. and G.W. developed tools, G.W. and S.S.
wrote the manuscript.
Additional information
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Welzel, G., Seitz, D. & Schuster, S. Magnetic-activated cell sorting
(MACS) can be used as a large-scale method for establishing zebrafish neuronal cell
cultures. Sci. Rep. 5, 7959; DOI:10.1038/srep07959 (2015).
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