Jurkat T cells were plated at a density of 2 × 105 cells per well. T20 (50 µM), the V-ATPase inhibitor bafilomycin A1 (100 nM, Sigma Aldrich, St. Louis, USA) or PBS were added 1 h prior to virus challenge. Cells were challenged with VSV-G HIV-1NL4-3ΔEnv and 4 h later, 200 µl of fresh culture medium were added. 48 h later, cells were fixed for 90 min in 4% PFA/PBS and HIV infection was monitored using an intracellular p24 staining (anti-p24 antibody, clone KC57-FITC, Beckmann Coulter, Brea, USA) in principle as reported32 (link).
Łyszkiewicz M., Ziętara N., Frey L., Pannicke U., Stern M., Liu Y., Fan Y., Puchałka J., Hollizeck S., Somekh I., Rohlfs M., Yilmaz T., Ünal E., Karakukcu M., Patiroğlu T., Kellerer C., Karasu E., Sykora K.W., Lev A., Simon A., Somech R., Roesler J., Hoenig M., Keppler O.T., Schwarz K, & Klein C. (2020). Human FCHO1 deficiency reveals role for clathrin-mediated endocytosis in development and function of T cells. Nature Communications, 11, 1031.
Corresponding Organization : Ludwig-Maximilians-Universität München
Other organizations :
Universität Ulm, Erciyes University, Medizinische Hochschule Hannover, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Tel Aviv University, University Hospital Carl Gustav Carus, University Hospital Ulm, German Center for Infection Research, German Red Cross
The V-ATPase inhibitor bafilomycin A1 (100 nM, Sigma Aldrich, St. Louis, USA)
dependent variables
HIV infection monitored using an intracellular p24 staining (anti-p24 antibody, clone KC57-FITC, Beckmann Coulter, Brea, USA)
control variables
Jurkat T cells plated at a density of 2 × 10^5 cells per well
Cells challenged with VSV-G HIV-1NL4-3ΔEnv
4 h later, 200 µl of fresh culture medium added
48 h later, cells fixed for 90 min in 4% PFA/PBS
negative control
PBS
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