Immunophenotyping of GPI-anchored proteins

HAP1 cells kindly provided by Thjin R. Brummelkamp (Netherlands Cancer Institute, Amsterdam, The Netherlands) were cultured in Iscove’s modified Dulbecco’s medium containing 10% fetal calf serum with necessary selection antibiotics [18 (link)]. Mouse monoclonal anti-CD59 (clone 5H8) and anti-DAF (clone IA10) were used as primary antibodies [22 (link)]. Phycoerythrin (PE)-conjugated goat anti-mouse IgG (Biolegend) was used as a secondary antibody. The bacterial pore-forming toxin proaerolysin and its variant fluorescent-labeled aerolysin (FLAER) were obtained from Protox Biotech [23 (link)].

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Rong Y., Nakamura S., Hirata T., Motooka D., Liu Y.S., He Z.A., Gao X.D., Maeda Y., Kinoshita T, & Fujita M. (2015). Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis. PLoS ONE, 10(9), e0138553.

Publication 2015

Phycoerythrin (PE)-conjugated goat anti-mouse IgG

Aerolysin Anti igg Antibiotics Antibodies Antibody Bacterial toxin Cancer Cd59 Cells Clone Cultured medium Fetal calf serum Goat Mouse Phycoerythrin Proaerolysin Protox

Corresponding Organization : Osaka University

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Variable analysis

independent variables

Cell line: HAP1 cells

dependent variables

Expression of CD59 and DAF proteins

control variables

Culture medium: Iscove's modified Dulbecco's medium with 10% fetal calf serum
Selection antibiotics

controls

Positive control: Mouse monoclonal anti-CD59 (clone 5H8) and anti-DAF (clone IA10) primary antibodies
Negative control: Not explicitly mentioned

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