Peripheral blood AKAP7 expression as an early marker for lymphocyte-mediated post-stroke blood brain barrier disruption

O’Connell, Grant C.; Treadway, Madison B.; Petrone, Ashley B.; Tennant, Connie S.; Lucke-Wold, Noelle; Chantler, Paul D.; Barr, Taura L.

Primary lymphocyte culture and adhesion assay. All cell culture was performed using aseptic technique under standard mammalian culture conditions. For isolation of primary lymphocytes, 10 mLs of whole peripheral blood was collected via EDTA vacutainer and peripheral blood mononuclear cells were isolated via density gradient centrifugation. Two rounds of differential plating were performed to separate monocytes from lymphocytes. Lymphocytes were expanded for 72 hours in a growth media comprised of RPMI-1640 (Life Technologies, Grand Island, NY) containing 10% autologous human serum, beta-mercaptoethanol (Gibco, Grand Island, NY), phytohaemagglutinin (PHA, Gibco), and HEPES (Gibco). For adhesion assays, expanded lymphocytes were suspended in serum-free media and plated in 6-well culture vessels coated in a matrix comprised of recombinant human collagen, fibronectin, and laminin (Gibco). After 20 minutes, non-adherent cells were collected from the culture supernatant via centrifugation and the remaining adherent cells were separately harvested via scraping. Cell fractions were lysed in Qiagen buffer RLT and stored at −80 for later RNA extraction. Adherent and non-adherent fractions from identical cultures were used for viability testing via trypan blue. Data represent lymphocyte populations obtained from three healthy donors, all assayed in triplicate.