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. 2014 Feb;88(3):1659–1672. doi: 10.1128/JVI.02014-13

FIG 2.

FIG 2

Lec1 cells are less susceptible to IAV infection than parental CHO cells. (A and B) CHO and Lec1 cells were incubated in medium alone (untreated; black histograms) or medium supplemented with 100 mU/ml of bacterial sialidase (sialidase; gray histograms), washed, and used in binding assays. (A) Binding of biotin-labeled MAA (i) or L-PHA (ii) was determined by flow cytometry. Representative histograms of triplicate samples are shown, and dashed lines represent cells stained with streptavidin-APC alone. MFI (± 1 SD) of binding to mock- or sialidase-treated cells for MAA (i) were the following: CHO, 31.2 ± 17.5 and 5.2 ± 3.6, respectively; Lec1, 35.6 ± 1.7 and 7.9 ± 1.4, respectively. MFI (± 1 SD) for L-PHA (ii) were the following: CHO, 13.6 ± 2.5 and 14.1 ± 1.4, respectively; Lec1, 1.4 ± 0.2 and 1.4 ± 0.2, respectively. (B) Binding of 5 μg/ml biotin-labeled BJx109 was determined by flow cytometry. Representative histograms of triplicate samples are shown, and dashed lines represent cells stained with streptavidin-APC alone. MFI (± 1 SD) for binding to mock- or sialidase-treated cells are shown (CHO cells, 395.3 ± 2.3 and 5.5 ± 0.1, respectively; Lec1 cells, 41.4 ± 4.2 and 3.7 ± 0.1, respectively.) (C and D) Monolayers of CHO or Lec1 cells were infected with 107 PFU of BJx109 at 37°C for 1 h as described in Materials and Methods. At 2 or 8 h, cells were fixed and stained for expression of viral NP or HA. (C) Representative images of CHO cells (i) and Lec1 cells (ii) using a Leica DMLB microscope (used to determine the percentage of IAV-infected cells) are shown. Images show NP or HA (FITC) and double-stranded nucleic acid (DAPI). (D) Fluorescent and total cells were counted in at least 4 random fields (>200 cells) of CHO and Lec1 cells at 8 h postinfection. Data show the mean percent infection (± 1 SD) and are representative of 5 independent experiments. Student's t test was used to determine significance (***, P ≤ 0.001). (E) Flow cytometry to determine expression of IAV NP at 2 h (i) or 8 h (ii) after infection with BJx109. Representative histograms from IAV-infected (white histograms) or mock-infected (gray histograms) triplicate samples and mean percentages of infected cells (± 1 SD) are shown. A gate was set to include 5% of cells in uninfected controls, and the percentage of infected cells was determined relative to this. Data are representative of 3 independent experiments.