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MB Sample ID: SA192475
| Local Sample ID: | OLGO_PP |
| Subject ID: | SU002129 |
| Subject Type: | Cultured cells |
| Subject Species: | Rattus norvegicus |
| Taxonomy ID: | 10116 |
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Collection:
| Collection ID: | CO002122 |
| Collection Summary: | RGC cell isolation. Primary retinal ganglion cells (RGCs) were isolated according to the two-step immunopanning protocol described in a previous study (Dvoriantchikova, Degterev et al., 2014). Briefly, whole retinas of 10 postnatal day 11-12 pups were incubated in papain solution (16.5 U/mL; Worthington Biochemical, LS003127) for 30 minutes. Macrophaged and endothelial cells were removed from the cell suspension by panning with anti-macrophage antiserum (Accurate Chemical, AIA31240). RGCs were bound to the panning plates containing the antibody against Thy1.2 (VWR, 102646-060) and were then released by incubation with trypsin solution (Sigma, T9935). RGCs were plated in a plate coated with poly-d-lysine (Sigma, P6407) and Laminin (Sigma, L-6274). RGCs were either not treated (addition of RGC growth media), treated with LPC 18:0 at 10 µM in RGC growth media, or treated with LPC 18:1 at 10 µM in RGC growth media. RGCs were incubated for 24 hours and harvested the next day. OPC cell culture. Oligodendrocyte Precursor Cells (OPC) were purchased from ScienceCell Research Laboratories (R1600, Carlsbad, CA 92008) and cultured according to manufacturer’s recommendations. In brief, plates were coated with poly-d-lysine (Sigma, P6407; 2 µg/cm2) and incubated overnight at 37ºC. OPC growth media was prepared using OPC media (ScienceCell Research Laboratories, 1601) supplemented with OPC growth supplement (ScienceCell Research Laboratories, 1652) and penicillin/streptomycin solution (ScienceCell Research Laboratories, 0503). OPC differentiation medium was prepared using OPC media (ScienceCell Research Laboratories, 1601) supplemented with OPC differentiation supplement (ScienceCell Research Laboratories, 1672), fetal bovine serum (ScienceCell Research Laboratories, 0005) and penicillin/streptomycin solution (ScienceCell Research Laboratories, 0503). Poly-d-lysine coated plate was washed twice with sterile water, OPC growth media was added to each well and plate was placed in incubator to equilibrate for 15 minutes (37ºC and 5% CO2). Frozen vial containing OPCs was warmed in 37ºC water bath and gently rotated to equally suspend cells. 15,000 cells/cm2 were seeded and left overnight (~16 to ~18 hours) in incubator. Media was changed daily for 2 days. OPCs were either not treated (addition of OPC growth media), differentiated (addition of OPC differentiation media), treated with LPC 18:0 at 10 µM in OPC growth media, or treated with LPC 18:1 at 10 µM in OPC growth media. OPCs were incubated for 24 hours and harvested the next day. OPCs and oligodendrocytes were further validated using mRNA analysis following published methods (Jäkel, Agirre et al., 2019). |
| Sample Type: | Cultured cells |
