Author: Qiao, Hui; Pelletier, Sandra L.; Hoffman, Lucas; Hacker, Jill; Armstrong, R. Todd; White, Judith M.
Title: Specific Single or Double Proline Substitutions in the “Spring-loaded” Coiled-Coil Region of the Influenza Hemagglutinin Impair or Abolish Membrane Fusion Activity Document date: 1998_6_15
ID: 78fjem8s_24
Snippet: Red blood cells (RBCs) were labeled with octadecylrhodamine (R18) or calcein AM (Molecular Probes, Inc., Eugene, OR) as described previously (Kemble et al., 1994) except that calcein AM was used at 10 M. Monolayers of cells expressing wt-or mutant HA0s were washed twice with RPMI media and (to enhance RBC binding) incubated with RPMI containing 0.1 mg/ml neuraminidase (Sigma Chemical Co.) for 1 h at 37 Њ C. Cells were then treated with trypsin t.....
Document: Red blood cells (RBCs) were labeled with octadecylrhodamine (R18) or calcein AM (Molecular Probes, Inc., Eugene, OR) as described previously (Kemble et al., 1994) except that calcein AM was used at 10 M. Monolayers of cells expressing wt-or mutant HA0s were washed twice with RPMI media and (to enhance RBC binding) incubated with RPMI containing 0.1 mg/ml neuraminidase (Sigma Chemical Co.) for 1 h at 37 Њ C. Cells were then treated with trypsin to cleave HA0 as described above. R18-or calcein AM-labeled RBCs, at ‫Ù‬ 0.05% vol/vol for COS 7 cells and 0.03% for 293T cells, were added to the HA-expressing cells for 20 min at RT, and unbound RBCs were removed by repeated washing. Cell monolayers were then incubated at 37 Њ C with pH 5 fusion buffer (10 mM MES, 10 mM Hepes, 120 mM NaCl, 10 mM succinate, and 2 mg/ml glucose) for 2 min (or as indicated), neutralized in the same buffer at pH 7, and observed with a fluorescence microscope. In specified experiments, the pH was adjusted as indicated.
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