Isolation and Analysis of Midbrain Mononuclear Cells

Mononuclear cells were isolated 4 weeks post-transduction from ventral midbrains with bilateral AAV injections, according to published protocols [27 (link), 30 (link)]. Briefly, midbrains were digested with 1 mg/mL Collagenase IV (Sigma) and 20 μg/mL DNAse I (Sigma) diluted in RPMI 1640 with 10% heat inactivated fetal bovine serum, 1% L-glutamine (Sigma), and 1% Penicillin-Streptomycin (Sigma). Mononuclear cells were separated out using a 30/70% Percoll gradient, as previously described [30 (link)]. Isolated cells were blocked with anti-Fcy receptor (clone 2.4G2 BD Biosciences) then incubated with fluorescent-conjugated antibodies against CD45 (clone 30-F11, eBioscience), CD11b (clone M1/70, BioLegend), MHCII (M5/114.15.2, BioLegend), Ly6C (clone HK 1.4, BioLegend), CD4 (clone GK1.5, BioLegend), and CD8a (clone 53-6.7, BioLegend). A fixable viability dye was used to distinguish live cells from debris per manufacturer’s instructions (Fixable Near-IR LIVE/DEAD Stain Kit, Invitrogen). Samples were analyzed using an Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo software (Tree Star).

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Williams G.P., Schonhoff A.M., Jurkuvenaite A., Thome A.D., Standaert D.G, & Harms A.S. (2018). Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson’s disease. Journal of Neuroinflammation, 15, 244.

Publication 2018

Collagenase IV DNAse I L-glutamine Penicillin-Streptomycin CD45 (clone 30-F11, CD11b (clone M1/70, MHCII (M5/114.15.2, Ly6C (clone HK 1.4, CD4 (clone GK1.5, CD8a (clone 53-6.7, Attune Nxt flow cytometer

Cd11b Cells Clone Collagenase Dnase Fetal bovine serum Fluorescent antibodies Glutamine Midbrains Ml iv Penicillin Percoll Stain Streptomycin Tree

Corresponding Organization :

Other organizations : University of Alabama at Birmingham

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Variable analysis

independent variables

AAV injections into the ventral midbrains

dependent variables

Characteristics of mononuclear cells isolated from the ventral midbrains 4 weeks post-transduction
Levels of CD45, CD11b, MHCII, Ly6C, CD4, and CD8a expression in the isolated mononuclear cells

control variables

Collagenase IV concentration (1 mg/mL)
DNAse I concentration (20 μg/mL)
RPMI 1640 medium with 10% heat inactivated fetal bovine serum, 1% L-glutamine, and 1% Penicillin-Streptomycin
Percoll gradient (30/70%)
Blocking with anti-Fcy receptor (clone 2.4G2 BD Biosciences)
Fluorescent-conjugated antibodies against CD45, CD11b, MHCII, Ly6C, CD4, and CD8a
Fixable viability dye to distinguish live cells from debris
Flow cytometry analysis using Attune Nxt flow cytometer and FlowJo software

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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