Lab tek 2 8 well chamber slide

Primary mixed glial cell cultures were prepared from the brains of postnatal 3–5-day-old (P3–P5) B6 mice, as previously described [12 (link)]. Astrocytes were prepared after removal of CD11b⁺ cells using Dynabeads (Dynal) conjugated with anti-CD11b Ab. The purity of astrocytes was >96% as determined by anti-GFAP and anti-CD11b immunofluorescence. Astrocytes were then seeded into culture flasks, 96-well plates, or Lab-Tek II 8-well chamber slides (Thermo Fisher Scientific, Waltham, MA, USA) at a density of 7.5 × 10⁶ cells per flask, 2.5 × 10⁴ cells per 96-well, or 1.0 × 10⁵ cells per chamber slide well and cultured in the medium described above for 5 days to form a confluent monolayer. LN⁻ cells were seeded on astrocytes at a density of 1.5 × 10⁶ cells per flask or 0.5 × 10⁴ cells per 96-well and chamber slide well and cultured for 7 days.

Cells were seeded onto Lab-Tek II 8-well chamber slides (Thermo Fisher Scientific, USA) and allowed to form a confluent monolayer. A 200 µl pipette tip was used to scratch the cell monolayer in each well. Cells were imaged at 10 × magnification using a Nikon Eclipse Ti-2 Inverted Microscope then incubated under standard conditions. Cell migration into the scratched area was documented over 48 hours. Images were analysed using the measuring tools in Fiji Image J. Cell migration at 48 hours was calculated by subtracting the width of the scratch at 48 hours from the width of the scratch at 0 hours. Cell migration was then divided by the width of the scratch at 0 hours to determine the percentage closure.

HeLa cells (parental cells or cells expressing His₁₀–SUMO2) were seeded in Lab-TekII 8-well chamber slides (Thermo Fisher Scientific, 154534). After treatment with TAK243, ML-792, or CHX, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 1% Triton X-100 for 15 min at room temperature, and then rinsed twice with PBST (PBS with 0.05% Tween 20). Immunostaining was performed with anti-SUMO2/3 antibody (Abcam, ab-81371 (8A2), 1:100) or anti-PML antibody (Bethyl, A301-167A, 1:100). The secondary antibodies were Alexa Fluor 488-conjugated anti-mouse secondary antibody (Life Technologies, Inc., A-11001, 1:500) and Alexa Fluor 555-conjugated anti-rabbit secondary antibody (Life Technologies, Inc., A-21428, 1:500). Nuclei were counter-stained with DAPI in the mounting media (Life Technologies, Inc., P36966). Images were taken with a Nikon Lucille spinning-disk confocal microscope equipped with a Hamamatsu ORCA-ER–cooled CCD camera (confocal, for 488- and 555-nm channels, and widefield, for the DAPI channel) and a ×100/1.4 oil objective at room temperature, remotely controlled with MetaMorph image acquisition software.