Table 2.
ECM coating conditions used in this study.
| Abbreviation | ECM components | References | Cost(€)/ml* |
|---|---|---|---|
| POL | 20 μg/ml Poly-L-ornithine + 20 μg/ml Laminin | 9 | 5.15 |
| POLF |
20 μg/ml Poly-L-ornithine + 20 μg/ml Laminin + 10 μg/ml Fibronectin |
10 | 8.23 |
| POLFM | 20 μg/ml Poly-L-ornithine + 20 μg/ml Laminin + 10 μg/ml Fibronectin + Matrigel (1:20) | 12 | 11.38 |
| POM | 20 μg/ml Poly-L-ornithine + Matrigel (1:50) | 11 | 1.79 |
| PEI | 0.1% Polyethyleneimine in borate buffer | 0.11 |
* The detailed calculation is listed in Table S1.
POL: 100 µg/ml Poly-L-ornithine (P4957, Sigma) was diluted to 20 µg/ml in Dulbecco’s Phosphate Buffered Saline (DPBS, D8662, Sigma). The culture ware was coated with 20 µg/ml Poly-L-ornithine at 4℃ overnight. Laminin at 1 mg/ml (L2020, Sigma) was thawed at 4℃ overnight and diluted to 20 µg/ml in DPBS. On the second day of coating, Poly-L-ornithine was removed, and the plates were rinsed with sterile water twice and then coated with 20 µg/ml of Laminin at 37 °C for 2 h. The coating matrix was removed and rinsed once with PBS before plating cells.
POLF: The dishes were first coasted with poly-l-ornithine as described above. The 1 mg/ml laminin and 1 mg/ml fibronectin (F1141, Sigma) stocks were pre-thawed at 4℃ and diluted with DPBS to contain 20 μg/ml laminin and 10 μg/ml fibronectin. Then poly-l-ornithine pre-coated plates were coated with laminin/fibronectin solution at 37 °C for 2 h. The coating matrix was removed and rinsed once with PBS before plating cells.
POLFM: After poly-l-ornithine, laminin and fibronectin coating as described above, Matrigel was thawed at 4℃ and diluted 20 × in KnockOut™ DMEM (10,829,018, Gibco), and then added to the same wells. The plates were kept at 37 °C for 2 h, and Matrigel was removed before use.
POM: After overnight coating of poly-l-ornithine, the same wells were coated with Matrigel (1:50) at 37 °C for 2 h, and Matrigel was removed before use.
0.1% PEI Solution: 50% PEI (P3143, Sigma) was diluted to 10% with sterile water as recommended by FujiFilm Cellular Dynamics, Inc. (https://www.fujifilmcdi.com/icell-motor-neurons-01279-gmnc01279). The 0.1% PEI working solution was freshly made by diluting 10% PEI at a 1: 100 ratio with 1 × borate buffer, which was made by diluting 20 × borate buffer (28,341, Thermo Scientific) with sterile water. The 0.1% PEI was filtered with a 0.22 μM sterile filter (A16534K, Lennox) before use.