Enzymatic treatment of reticulocyte-enriched samples was performed by incubating 10 µl of the cell suspension with 1 mg/ml trypsin (from bovine pancreas, Sigma), 1 mg/ml chymotrypsin (from bovine pancreas, Sigma) and 0.5 U/ml neuraminidase (from Vibrio cholerae, Sigma) at 37 °C for 1 hour69 (link). CR1 enzymatic cleavage efficiency was tested by FACSCalibur 4-color flow cytometer (BD Biosciences)15 (link) and neuraminidase treatment was assessed by performing an agglutination test using lectin from the peanut Arachis hypogaea, to detect T-antigen which is exposed after sialic acid cleavage69 (link).
Prajapati S.K., Borlon C., Rovira-Vallbona E., Gruszczyk J., Menant S., Tham W.H., Kattenberg J.H., Villasis E., De Meulenaere K., Gamboa D., Vinetz J., Fujita R., Xuan X.N., Urbano Ferreira M., Niño C.H., Patarroyo M.A., Spanakos G., Kestens L., Abbeele J.V, & Rosanas-Urgell A. (2019). Complement Receptor 1 availability on red blood cell surface modulates Plasmodium vivax invasion of human reticulocytes. Scientific Reports, 9, 8943.
Other organizations :
Instituut voor Tropische Geneeskunde, Walter and Eliza Hall Institute of Medical Research, Universidad Peruana Cayetano Heredia, Universidad de San Martín de Porres, National Institute of Malariology, Parasitology and Entomology, Universidade de São Paulo, Fundación Instituto de Inmunología de Colombia, Hellenic Center for Disease Control & Prevention
Enzymatic treatment of reticulocyte-enriched samples by incubating with trypsin, chymotrypsin, and neuraminidase
dependent variables
CR1 enzymatic cleavage efficiency
Neuraminidase treatment assessed by agglutination test using peanut lectin to detect T-antigen
control variables
Cell suspension volume (10 µl)
Trypsin concentration (1 mg/ml)
Chymotrypsin concentration (1 mg/ml)
Neuraminidase concentration (0.5 U/ml)
Incubation temperature (37 °C)
Incubation duration (1 hour)
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