Repository of the University of Rijeka

Ban, Jelena

SUPPLEMENTARY INFORMATION 
 
An improved method for growing neurons: comparison 
with standard methods 
Pozzi D.1 ,⃰ Ban J.1,2 ,⃰ Iseppon F.1 and Torre V1. 
1Neurobiology Sector, International School for Advanced Studies (SISSA), via Bonomea 265, 
34136 Trieste, Italy.  
2Department of biotechnology, University of Rijeka, Radmile Matejčić 2, 51000 Rijeka, Croatia.  
  ⃰These authors contribute equally to this work.  
Correspondence and requests for materials should be addressed to V.T. (email: torre@sissa.it).  
 
 
Supplementary Figure 1. Elaborated growth cone morphology in Neurobasal/B27-based media. 
Hippocampal culture at DIV1 in B27 (A) and ACM (B) stained for F-actin (green) and β-tubulin III 
(TUJ1, red). Such complex morphology was never observed in FBS-based medium at DIV1. Scale 
bar, 5µm.  
 
  
 
 
 
Supplementary Figure 2. Glial cell morphology at DIV1. (A) Hippocampal culture in FBS (left), 
B27 (middle) and ACM (right) stained for F-actin (green) and Hoechst nuclear stain (blue). (B) 
Fluorescent confocal images of the same fields in A but for neuronal marker β-tubulin III (TUJ1, red) 
and Hoechst nuclear stain (blue). Glial cells are identified as F-actin-positive and β-tubulin III- 
negative cells. Scale bar, 25 µm. 
 
 Supplementary Figure 3. Glial cells morphology after two weeks of culture. (A) Hippocampal 
culture at DIV14 in FBS (left), B27 (middle) and ACM (right) stained for glial fibrillary acidic protein 
(GFAP). Scale bar, 50 µm. (B) Astrocytes processes at higher magnification. Corresponding insets 
are indicated in A. Scale bar, 5 µm. 
 
Supplementary Figure 4. The blocking action of TTX: the neuronal origin of calcium transients in 
a representative calcium imaging experiment on cells grown in FBS medium for 2 months and loaded 
with 4 µM Oregon Green 488 BAPTA - 1® calcium indicator. Cells analyzed in (A) were identified 
as neurons because their spontaneous calcium transients (B) were blocked by the addition of 1 µM 
TTX, a well-known specific antagonist of voltage gated sodium channels (C).  
  
Supplementary Video 1. Fluorescent images of neuronal cultures grown for 1.5 months in ACM, 
loaded with 4 µM Oregon Green 488 BAPTA - 1® and imaged with an EM –CCD camera at a speed 
of 7 Hz for 10 min.  
 
 Neuronal medium (MEM 
w/Glutamax® – based) 
Neurobasal/B27 
(Neurobasal® - based) 
Amino Acids Concentration (mg/l) Concentration (mg/l) 
L – Arginine hydrochloride 126.0 84.0 
L - Cysteine 24.0 31.5 
L-Histidine hydrochloride-H2O 42.0 42.0 
L - Isoleucine 52.0 105.0 
L - Leucine 52.0 105.0 
L – Lysine hydrochloride 73.0 146.0 
L - Methionine 15.0 30.0 
L - Phenylalanine 32.0 66.0 
L - Threonine 48.0 95.0 
L - Tryptophan 10.0 16.0 
L - Tyrosine 36.0 72.0 
L - Valine 46.0 94.0 
L – Alanyl - Glutamine 406.0 108.5 
Glycine  30.0 
L - Alanine  2.0 
L – Asparagine – H2O  0.83 
L - Proline  7.76 
L - Serine  42.0 
   
Vitamins   
Choline chloride 1.0 4.0 
D – Calcium pantothenate 1.0 4.0 
Folic Acid 1.0 4.0 
Niacinamide 1.0 4.0 
Pyridoxal hydrochloride 1.0 4.0 
Thiamine hydrochloride 1.0 4.0 
Riboflavin 0.1 0.4 
i- Inositol 2.0 7.2 
Vitamin B12 1.5 0.0068 
   
Inorganic salts   
Calcium chloride 264.0 200.0 
Sodium Bicarbonate 2200.0 2200.0 
Sodium chloride 6800.0 4000.0 
Sodium phosphate monobasic 158.0 125.0 
Magnesium sulfate 200.0  
Ferric nitrate  0.1 
Magnesium chloride  77.3 
Potassium Chloride 400.0 400.0 
Zinc sulfate  0.194 
   
Other components   
D - glucose 7000.0 4500.0 
Phenol red 10.0 8.1 
HEPES 3600.0 2600.0 
Sodium Pyruvate  25.0 
Apo - transferrin 100 B27 
Biotin 0.1 B27 
Insulin 30.0 B27 
 
Supplementary Table 1. Chemical composition of Minimum Essential Medium (MEM, 
cat.no.41090028)-based Neuronal medium and Neurobasal medium (cat.no.21103049, both from 
ThermoFisher). The two media differ in terms of concentration and number of components. Most of 
the amino acids and vitamins are more concentrated in Neurobasal comparing to MEM, while 
inorganic salts in MEM are less numerous than in Neurobasal but more concentrated. Note that 
components such as apo – transferrin, biotin and insulin were added at specific concentration to the 
neuronal medium formulation supplemented with FBS, while in Neurobasal/B27 they are included in 
the commercial B27 supplement at unknown concentration (for complete composition list of the B27 
supplement please see cat.no.17504044, Thermo Fisher Scientific). In both cases, the antibiotic 
gentamycin was added at a concentration of 2.5 µg/l.  
 


Appendix A. Supplementary data

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Appendix A. Supplementary data

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