C-gly-Cn and E-gly-Cn (100 μg) were incubated with 107 GFP Hc yeasts for 1 h at 37 °C in PBS. Hc yeasts incubated in PBS alone were used as a control. Following incubation, cells were washed (3X) with PBS to remove unbound gly and enumerated using a haemocytometer. Gly incorporation by Hc GFP yeast was determined by FACS analysis using mAb 2D10 as described above.
Hc were also suspended at 107 cells/mL in HAM F-12 medium. Next, 50 μL (106 total yeast) was added to individual wells of polystyrene 96-well plates (Fisher, MA) and incubated with 10μg of C-gly-Cn or E-gly-Cn in 50 μL of HAM F-12 media). Plates were incubated at 37 °C without shaking for 48 h. Pellicle formation was assessed as described previously.
To compare the relative incorporation of Hc G217B with the high α-1, 3-glucan strain Hc G186A, yeasts were incubated with the Cn gly fractions for different time intervals and incorporation was detected by indirect ELISA as described58 (link).
Hc were also suspended at 107 cells/mL in HAM F-12 medium. Next, 50 μL (106 total yeast) was added to individual wells of polystyrene 96-well plates (Fisher, MA) and incubated with 10μg of C-gly-Cn or E-gly-Cn in 50 μL of HAM F-12 media). Plates were incubated at 37 °C without shaking for 48 h. Pellicle formation was assessed as described previously.
To compare the relative incorporation of Hc G217B with the high α-1, 3-glucan strain Hc G186A, yeasts were incubated with the Cn gly fractions for different time intervals and incorporation was detected by indirect ELISA as described58 (link).
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