Table 1:

Troubleshooting of procedure.

Step	Problem	Cause	Suggestion
1.2.	Failure of trypsinisation	Inhibitory FBS in residual media	Ensure entire media is washed away with HBSS before trypsin
2.1.	DMSO pools at centre of well & is toxic to the cells located at center	Dense DMSO sits at center of well when added directly to well	Add DMSO(-solubilized drugs) to media in tube, mix and add media/DMSO mixture to well
2.5.	Loss of formazan	Insoluble formazan crystals lost in MTT	Do not agitate plate or disturb formazan when removing MTT
3.3./13.2.	Hyper-exposed calcein-AM staining	Incubation of live cells with calcein-AM > 2 h	Ensure calcein-AM stained cells are imaged in < 2 h
3.5.	Underestimation of neurite length	Gridlines with too large distance between lines	T of 25 µm is recommended for SH-SY5Y cell neurite analysis
4./9./14.	Poor cell survival following transfection	Excessive time taken for electroporation protocol	Perform electroporation protocol in > 30 min
4./9./14.	Poor transfection efficiency & survival	Transfection protocol not optimized	See ‘Transfection of Cultured Neural Cells Optimization’
5./10.	Low protein yield	Excessive lysate dilution	Use 150 µl lysis buffer per well
5./10.	Absence of activated signaling pathway(s)	Phosphorylation of proteins lost	Use phosphatase inhibitors in lysis buffer
6.3.	Meninges on VM tissue	Inefficient removal of meninges from VM	Peel VM tissue from meninges. Do not attempt to cut meninges
7.4.	Unanalysable/misplaced neurons at edge of well	50 µl of plated cells spills to edges of well	Ensure 50 µl of plated cells remains at center of well
8.3./13.3.	Underestimation of neurite length	Gridlines with too large distance between lines	T of 50 µm is recommended for primary neuron growth analysis
9.4.	GFP-positive cells lost	Fixing can quench GFP	Use anti-GFP antibody
11.5.	Wrong ganglia dissected for cell culture	SCG & nodose ganglia similar & close together	SCG is distinctly larger than nodose ganglion
11.6.	Loss of dissected SCG	‘Sticky’ SCG stuck in pipettes &/or tubes	Use unplugged Pasteur pipette that can wash out with trypsin
3./4./8./9./13./14.	Incorrect visualization of target neurons	Inappropriate labeling/identification of neurons	See ‘Visualization of Cultured Neural Cells’