Focal demyelinated lesions of the corpus callosum of 8-12 week old
C57Bl6J male mice were dissected out and homogenized with a 2 ml dounce. A
Percoll (Sigma-Aldrich) gradient was used to isolate cells from myelin debris.
Samples were blocked with Fc-block [LEAF-purified anti-mouse CD16/32 (Biolegend,
101321)], then incubated with fluorochrome-conjugated antibodies CD11b-PeCy7
(eBioscience, 25-0112-82, 1:100) and CD45-BV605 (Biolegend, 103139, 1:100) for
30 minutes on ice, followed by incubation with ‘FITC Annexin-V apoptosis
detection kit with 7-AAD’ for 15 minutes at room temperature (Biolegend,
640922, 1:20). Following washes in buffer, samples were run on the BD LSR
Fortessa (6 laser) analyser, and analysed using FlowJo version 9/10 software
(FlowJo LLC).
C57Bl6J male mice were dissected out and homogenized with a 2 ml dounce. A
Percoll (Sigma-Aldrich) gradient was used to isolate cells from myelin debris.
Samples were blocked with Fc-block [LEAF-purified anti-mouse CD16/32 (Biolegend,
101321)], then incubated with fluorochrome-conjugated antibodies CD11b-PeCy7
(eBioscience, 25-0112-82, 1:100) and CD45-BV605 (Biolegend, 103139, 1:100) for
30 minutes on ice, followed by incubation with ‘FITC Annexin-V apoptosis
detection kit with 7-AAD’ for 15 minutes at room temperature (Biolegend,
640922, 1:20). Following washes in buffer, samples were run on the BD LSR
Fortessa (6 laser) analyser, and analysed using FlowJo version 9/10 software
(FlowJo LLC).