Milliplex mouse cytokine magnetic kit

The V-SVZ was collected as described above. Each sample contained tissue pooled from 3 to 6 pups. Tissue was weighed to determine the appropriate volume of lysis buffer (20mM Tris-HCl, 250mM NaCl, 0.05% Tween 20, complete Mini Protease Inhibitor Cocktail EASYpacks tablet (1 tablet for 10mL of lysis buffer) (Roche, Cat#04693124001)) and manually homogenized, then centrifuged at 14K x g for 10 min at 4°C. The supernatant was removed into a new tube and stored at −80°C until analysis. Analysis was performed using the Milliplex Mouse Cytokine Magnetic Kit (EMD Millipore, Cat#MCYTOMAG-70K) and Luminex MAGPIX instrument at the Vanderbilt Hormone Assay and Analytical Service Core. The lysis buffer was used as the matrix solution in the background, standard curve, and control wells.

The right and left dorsal V-SVZ were collected from P7 pups according to regional delineations established previously, specifically regions ii/iiiC and ii/iiiD^{72 (link)}. Each sample contained tissue pooled from 3–6 pups. Tissue was weighed to determine the appropriate volume of lysis buffer (20mM Tris-HCl, 250mM NaCl, 0.05% Tween-20, cOmplete^™ Mini Protease Inhibitor Cocktail EASYpacks tablet (1 tablet for 10mL of lysis buffer) (Roche, Cat # 04693124001)) and manually homogenized, then centrifuged at 14K × g for ten minutes at 4°C. The supernatant was removed into a new tube and stored at −80°C until analysis. Analysis of six analytes (interferon γ (IFNγ), interleukin 1β (IL-1β), IL-6, IL-10, IL-17, and tumor necrosis factor α (TNFα)) was performed using the Milliplex Mouse Cytokine Magnetic Kit (EMD Millipore, Cat # MCYTOMAG-70K) and Luminex MAGPIX instrument at the Vanderbilt Hormone Assay and Analytical Service Core. The lysis buffer was used as the matrix solution in the background, standard curve, and control wells.