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Feng et al. Growth cone collapse in adult sensory neurons

The EG were bathed in DMEM/F12 medium (Invitrogen EG-conditioned medium or normal supplemented
11330-032) supplemented with 20% FBS and 1% P/S. neurobasal medium for the comparison groups
EG from passages 6-10 were acclimated to DMEM/ containing 200 ng/mL SEMA3A to obtain a bath
F12 containing 2% FBS and 1% P/S. Twenty-four concentration of 100 ng/mL. The medium in the
hours later, cells were rinsed in phosphate buffered treatment groups also contained either anti-NGF, anti-
saline (PBS, pH 7.35) and subsequently bathed in BDNF, anti-GDNF, anti-NT-3 added in PBS to achieve
supplemented neurobasal medium. After another 24 h, a well concentration of 2.5 µg/mL. Equal volumes of
the medium was conditioned and centrifuged for 5 min PBS were added to control wells. The cultures were
at 4,000 g to remove any particulates. incubated for 1 h and then fixed and stained.

Collapse assay Post-treatment
A collapse assay was adapted from Reza et al. Similar to the previous experiments, half of the
[16]
and experimental conditions were modified from medium in each well was replaced with fresh
Hansebout et al. EG-conditioned medium was neurobasal medium containing 200 ng/mL SEMA3A
[24]
administered before, during or after SEMA3A (R&D to obtain a bath concentration of 100 ng/mL. After
System 1250-S3) mediated collapse (pre-treatment, a 40-min incubation, 2.5 mL of the bathing medium
co-treatment, post-treatment). Each time setting was was replaced with either EG-conditioned medium
assessed using serial assays. To test individual growth with SEMA3A or supplemented neurobasal medium
factor involvement, antibodies to one of NGF, BDNF, with SEMA3A. Pilot studies revealed that 1.5 mL of
GDNF or NT-3 were added to select wells. Each EG medium was inadequate to prevent collapse after
neurotrophic factor was investigated with or without SEMA3A administration in our model. Also added
conditioning media (+/-CDN) and with or without was either anti-NGF, anti-BDNF, anti-GDNF, anti-
antibody (+/-aB). The negative (model; -CDN/-aB) and NT-3 in PBS for a final concentration of 2.5 ng/mL or
positive (+CDN/-aB) control groups show the effect of equal volumes of PBS for controls. These cells were
enteric glia on SEMA3A-mediated collapse before the incubated for another 20 min for a total 1-h incubation
addition of the antibodies. Cells were incubated at 37°C period before fixing and staining.
and PBS was used as a vector control for antibodies in
each experiment arm. The final working concentration Staining
of SEMA3A in all of the wells was 100 ng/mL. The final Phalloidin staining, which specifically binds to F-actin,
antibody concentration in each well was 2.5 µg/mL. All was used to visualize the actin-dense cytoskeleton of
cells were later fixed in 4% perfluoroalkoxy alkanes the growth cones. [31,32] Specific methods were modified
(PFA) containing 10% sucrose and subsequently from Hansebout et al. After a 10-min fixation in a 4%
[24]
stained. The detailed procedures are as follows: PFA and 10% sucrose solution, the cells were washed
in PBS, permeabilized with 0.05% Triton X-100 for 5
Pre-treatment min, and then treated with 1% bovine serum albumin
Half of the medium in each well was replaced with for 30 min to reduce background staining. The cells
EG-conditioned medium or normal supplemented were then stained for 1 h with Alexa-488 phalloidin
neurobasal medium for positive and negative controls (Invitrogen A12379) to visualize the growth cones
respectively. Each EG-conditioned and control and cell morphology and then counter-stained with
neurobasal group also received either anti-NGF Propidium iodide (Sigma) to visualize the nuclei. After
(RD System AMK0208091), anti-BDNF (Millipore a double wash in PBS and a final wash in double-
AB15130P), anti-GDNF (RD System AFW0408071), distilled water, coverslips were mounted onto glass
or anti-NT-3 (Chemicon AB1780SP) dissolved in PBS slides using VectaShield mounting medium (Vector
to reach a working concentration of 2.5 µg/mL while Laboratories H-1,000), and edges of the coverslips
controls were given PBS in equal volumes. After a were sealed with clear nail polish.
1-h incubation, all cultures were then treated with
100 µg/mL SEMA3A in PBS (R&D Systems 1250-S3- Data gathering
025) to obtain a bath concentration of 100 ng/mL as The ratio of collapsed to uncollapsed growth cones
per Wanigasekara et al. and Reza et al. One hour was measured as per Kapfhammer et al. [33] and
[18]
[16]
later, cells were fixed and stained. Brown et al. [34] noting the approximate cell body size
of each neurite, so as to exclude large-diameter DRG
Co-treatment neurons that have been shown to be unresponsive
This experiment was almost identical to the previous to SEMA3A. [16] Since the study used isolated DRG
one with the exception of the timing of the treatment. from adult rats rather than explanted embryonic DRG
Half of the medium in each well was replaced with as used in the study by Kapfhammer et al. [33] growth
182 Neuroimmunology and Neuroinflammation ¦ Volume 3 ¦ August 31, 2016