Proteoextract

To remove immunoglobulins, a combination of a commercially available IgG removal column utilizing Protein A (ProteoExtract, Merck KGaA, Darmstadt, Germany) and manual depletion with Sepharose G beads (Millipore Sigma) was performed as previously described.³⁵ Serum samples collected on day 35 from 6 MHC mismatched recipients (3, A5a recipients; 3, A3b recipients) were immunoglobulin depleted. Briefly, 100 μL of serum was added to 900 μL of 1× Binding Buffer. The sample was passed through the IgG removal column in a dropwise manner. Three hundred microliter of undiluted eluate was then combined with 200 μL of preconditioned Protein G Sepharose beads and incubated at 20°C for 1 hour with gentle mixing. After 1 hour, the samples were centrifuged at 4000g for 5 minutes and the immunoglobulin depleted supernatant collected. After sample processing, 2 mL of Protein A Elution Buffer was passed through the IgG removal column and collected. Microcytotoxicity assays were repeated as above with respective donor PBLs being combined with recipient serum diluted to a 1:10 dilution with 1× Binding Buffer, recipient immunoglobulin depleted serum, or recipient serum IgG removal column eluate.

Astrocytes grown on dishes were washed three times with PBS, and the membrane fraction was isolated using ProteoExtract (Merck Millipore, Darmstadt, Germany). Total protein was determined using the BCA assay. Each sample (30 μg per well) was separated using SDS-PAGE and subsequently transferred onto polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). The membranes were incubated with mouse CD55-antibody (Acris Antibodies, Herford, Germany) or rabbit CD59-antibody (Proteintech Group, Rosemont, USA) as primary antibodies for 8 h and then with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (DakoCytomation, Glostrup, Denmark). The bands were detected using an enhanced chemiluminescent detection system (ECLPlus; GE healthcare, Tokyo, Japan) with an image analyzer (LAS-3000; Fuji Photo FilmJapan, Kanagawa, Japan).