Pure OPC (POPC) cultures were generated from male and female WT P5-9 mice as previously described (45 (link)). At day 9 in culture, PDGFαα and NeuroTrophin 3 (NT3) were withdrawn to allow oligodendrocyte differentiation and cells were stimulated with 5% Treg-CM, 5% CCN3-depleted Treg-CM, 5% IgG isotype-depleted Treg-CM, or controls for up to 3 d. Purity was assessed in random cultures at 3–5 d after setup and was over 93%.
POPCs were fixed directly in 4% PFA (pH 7.4) (Sigma) for 15 min at RT. POPCs were blocked in 10% NGS (Vector Laboratories s-1000) with 0.1% Triton X-100 in PBS for 1 h. Cells were then incubated with primary antibodies for Olig2 (1:400 Millipore AB9610) and MBP (1:1,000 Millipore MAB386) in 2% NGS with 0.01% Triton X-100 in PBS overnight at 4 °C. Afterward, OPCs were incubated with secondary antibodies (1:1,000, goat anti-rabbit 488, A11034 and 1:1,000, goat anti-rat 594, A11007, both Life Technologies) for 1 h at RT. Cells were stained with DAPI for 5 min at RT. Immunofluorescence was detected using an EVOS FL microscope at 10× magnification and n = 5 wells per condition.
POPCs were fixed directly in 4% PFA (pH 7.4) (Sigma) for 15 min at RT. POPCs were blocked in 10% NGS (Vector Laboratories s-1000) with 0.1% Triton X-100 in PBS for 1 h. Cells were then incubated with primary antibodies for Olig2 (1:400 Millipore AB9610) and MBP (1:1,000 Millipore MAB386) in 2% NGS with 0.01% Triton X-100 in PBS overnight at 4 °C. Afterward, OPCs were incubated with secondary antibodies (1:1,000, goat anti-rabbit 488, A11034 and 1:1,000, goat anti-rat 594, A11007, both Life Technologies) for 1 h at RT. Cells were stained with DAPI for 5 min at RT. Immunofluorescence was detected using an EVOS FL microscope at 10× magnification and n = 5 wells per condition.
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