Figure 1.
Phase contrast images of DRG purification and myelination.
(A) DRG cultures at 24 h before adding antimitotic reagents cocktail, black arrow show DRG neurons, white arrowhead show non-neurons cells; (B) DRG cultures 72 h without high dose antimitotic reagent cocktail treatment, Schwann cell associated with neuritis (arrow) and other big flat non-neuron cells migrated out and grew between neuritis (arrowhead); (C) DRG cultures at 7 day without cocktail treatment, Schwann cell and other non-neuron cell formed a cellular lawn (white star) with DRG neurons sitting on them; (D) DRG cultures 72 h after cocktail treatment, abundant neurites and neurons were observed, dead non-neuron cell debris (black arrow) floating in the medium; (E) DRG cultures at 7 day after cocktail treatment, a pure DRG population without dead non-neuron cells established, single or small cluster of DRG neurons showed typical morphologies with no signs of damage, clean neurites without non-neuron cells (black star); (F) Cocktail treated DRG cultures 14 days after seeding Schwann cells and initiating myelination with ascorbic acid, abundant mature myelin had formed (arrow). Scale bar 50 µm.
Figure 2.
The survival of DRG neurons and non-neuronal cells after cocktail treatment.
(A–F) The immunocytochemistry for DRG neurons and non-neuron cells (on day 4). A–C: non-neuron cells (include all the S100+, P75 NRT+ and fibronect+ cells); Scale bar 10 µm; D–E: DRG neurons (include the NF-100+ and tuj 1+ cells); Scale bar 50 µm. F. The survival of DRG neurons and non-neuronal cells after cocktail treatment. Cell survival assessed as mean cells counts per well after 1, 7, 10 days culture. Data are means ± SEM. (n = 3). (Dunnett's test; P>0.05).
Figure 3.
DRG neurons viability (compared with traditional method).
Data are means ± SEM. (n = 5). No significantly difference was observed in cell viability between new method and traditional method. (Dunnett's test; P>0.05). At the end of 7 d culture period the vast majority of DRG neurons also remained viable, as evidenced by the high level of Trypan blue exclusion in all culture conditions.
Figure 4.
Neurite growth of purified DRG neurons.
Cells were fed with NGF on day 0, 2, 4, 6. (A–H) The representative phase contrast images of DRG neurons. A–D: Neurite growth of DRG neurons which purified by traditional method on day 1 (A), 3 (B), 5 (C), 7 (D). E–H: Neurite growth of DRG neurons which purified by new method on day 1 (E), 3 (F), 5 (G), 7 (H). I: The comparison of neurite growth of purified DRG neurons between traditional method and new method. Data are means ± SEM. (n = 9). No significantly difference was observed in neurite length between new method and traditional method. (Dunnett's test; P>0.05); Scale bar 50 µm.
Figure 5.
Myelin formation detected by immunofluorescence for myelin specific markers.
(A) Peripheral protein zero (P0, red), (B) Myelin basic protein (MBP, Green) and (C) Merge of the A and B and count staining with DAPI (blue) for nucleus. Abundant mature myelin and Ranvier node (arrow) were observed; Scale bar 50 µm. (D) Caspr (red), (E) Nav1.6 (green) and (F) Merge of the D and E. The formation of mature node of Ranvier in DRG-Schwann cell co-culture using one time high dose impact treatment were observed; Scale bar 5 µm.
Figure 6.
Schematic diagram of the experimental procedure of the new method comparison with tradition method.
(A) The new one time high dose impact treatment method, after 24 h in NG medium, DRG cultures were treated with NGF stock solution containing 20 µM FUDR plus 0.5 µM Ara C cocktail for 72 h, then switched to normal NG medium, at 7 day, a pure DRG neuron population (>99%) were obtained. At 10 day, these DRG neurons were ready for SC addition. (B) The classical multiple time treatment method, after 24 h in normal medium, DRG cultures were treated with NGF stock solution containing 10 µM FUDR for 48 h, then switched to NG medium for 48 h, this cycle was repeated at least 3 times; then DRG culture were maintained in NG medium for 1 week with 3 times medium changes to obtain the pure DRG population and these cultures were ready for Schwann cell addition.