2.7 Biopanning assays
The biopanning assays were carried out using established
protocols18,23. Tissue culture treated 6-well plates
(Corning) were coated with ≥300,000 g/mol Poly-D-Lysine (PDL)
(Sigma-Aldrich) by following the procedure described by the
manufacturer. Sterile water was used to dissolve the PDL powder to 0.1
mg/mL. Surfaces were coated with PDL solution at 1 mL/25
cm2, and allowed to incubate for 5 min. The solution
was then aspirated and plates were washed twice with sterile water and
then left to dry overnight. Mammalian cell monolayers were grown in the
PDL-coated 6-well plates to 70-100% confluency and washed three times
with PBSCMA (PBS containing 0.9 mM CaCl2, 0.49 mM
MgCl2, and 0.1% BSA). Induced yeast samples were washed
twice with PBSCMA and centrifuged at 3,500×g for 3 min. Yeast samples
were resuspended in PBSCMA at 5×107 yeast cells/mL. 1
mL of the yeast sample was added to each well (5×107yeast cells/well – an approximate yeast:mammalian cell ratio of 40:1
assuming that a confluent 6-well plate contains
≈1.2×106 cells (Thermo Fisher specifications)).
Co-incubation was carried out for 2 hr, after which five washes with
PBSCMA were conducted. Washes consisted of adding 1 mL of buffer to each
well and gently rocking the plate back and forth 25 times followed by
five rotations and then aspirating. Binding was detected via phase
contrast microscopy using an EVOS XL (Thermo Fisher Scientific) 40×
objective and quantified through manual counting of images in ImageJ.
Kinetic assays were conducted by varying the yeast/mammalian cell
incubation times at a fixed ratio of 40:1. Titrations were carried out
by varying the yeast:mammalian cell ratios while keeping the incubation
period constant at 2 hr.