Isolation and culture of sensory neurons
Dorsal root ganglia (DRGs) were harvested from naïve male Wistar rats
following their sacrifice by an overdose of isoflurane (5%) (AbbVie,
Wiesbaden, Germany). Immediately thereafter, the thoracic and lumbar
spinal regions were exposed and all DRGs were collected in a working
medium (DMEM/HAM’s F-12 without penicillin/streptomycin). 1.25%
collagenase was added just before incubation. Following their incubation
at 37°C for 60 min, DRGs were washed three times with PBS and incubated
in the working medium digestive solution with trypsin for 10 min at
37°C. Thereafter, the tissue was further triturated using plastic
pipette tips and filtered through a 40 µl filter. The filtrate was
centrifuged and the pellet was resuspended in 1 ml culture medium
(DMEM/HAM’s F12 supplemented with 1% penicillin/streptomycin and 10%
horse serum). Neurons were then seeded onto poly-L-lysine coated plastic
culture dishes (35 mm) and placed in an incubator (5%
CO2 at 37°C). One hour later, 2 ml of culture medium
were added to the cell culture and neurons were incubated for 24–48 h
until patch clamp recordings or calcium imaging, as previously described
(Nockemann et al., 2013; Dembla et al., 2017).