A human neuroglioma H4 cell-derived cell line stably over-expressing αSyn from a tetracycline inducible promoter20 (link) was grown in serum-free X-VIVO media (Lonza Group, Basel, Switzerland). Cells were seeded in 96-well plates at 100 K cells per well. The next day, compound was added along with 5 µg/ml tetracycline to induce αSyn overexpression. Cells were cultured overnight and the next day were fed 4 μM red fluorescent beads (In Vitrogen, Carlsbad, CA) for 90 minutes at a cell-to-bead ratio of 1:10. Plates were gently washed with 100 µl/well media twice, fixed and stained with HEMA3 (Thermo Fisher, Waltham, MA). Plates were dried overnight and read on an ArrayScan (Thermo Fisher, Waltham, MA). As the HEMA3 stain absorbs light, the internalized beads are less fluorescent than the outside beads. Tet/non-tet and 484228 samples were run on each plate.
Tóth G., Neumann T., Berthet A., Masliah E., Spencer B., Tao J., Jobling M.F., Gardai S.J., Bertoncini C.W., Cremades N., Bova M., Ballaron S., Chen X.H., Mao W., Nguyen P., Tabios M.C., Tambe M.A., Rochet J.C., Junker H.D., Schwizer D., Sekul R., Ott I., Anderson J.P., Szoke B., Hoffman W., Christodoulou J., Yednock T., Agard D.A., Schenk D, & McConlogue L. (2019). Novel Small Molecules Targeting the Intrinsically Disordered Structural Ensemble of α-Synuclein Protect Against Diverse α-Synuclein Mediated Dysfunctions. Scientific Reports, 9, 16947.
Other organizations :
Sunesis (United States), Laboratoire de Biophotonique et Pharmacologie, University of California, San Diego, Howard Hughes Medical Institute, University of California, San Francisco, University of Cambridge, Purdue University West Lafayette, University College London, Institute of Structural and Molecular Biology, Prothena (United States)
Tetracycline (5 µg/ml) to induce αSyn overexpression
dependent variables
Internalized fluorescent beads
control variables
Cell line (human neuroglioma H4 cell-derived cell line stably over-expressing αSyn from a tetracycline inducible promoter)
Serum-free X-VIVO media
Cell seeding density (100 K cells per well)
Fluorescent bead concentration (4 μM)
Cell-to-bead ratio (1:10)
Incubation time for fluorescent bead uptake (90 minutes)
Washing steps (2 washes with 100 µl/well media)
HEMA3 staining
positive control
Tet/non-tet samples
negative control
484228 samples
Annotations
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