2.10 Adherent cell-based enrichment
PD-L1- CHO-K1 and PD-L1+ CHO-K1
cells were grown to 90-100% confluency in tissue culture-treated 150 mm
petri dishes (Corning) that were PDL-coated. The adhered mammalian cells
were washed three times with PBSCMA and the induced yeast mock library
was washed twice. For the first round of selections,
1010 yeast cells were resuspended in 15 mL of PBSCMA
and added to PD-L1- CHO-K1 cells at a ratio of 550:1
yeast/mammalian cells for 30 min at 4°C (negative selection). The
supernatant was slowly collected from the plate and cells were washed
five times with PBSCMA, while collecting the supernatant for each wash.
The pooled supernatant was then spun down and resuspended in 15 mL of
PBSCMA and incubated with PD-L1+ CHO-K1 cells for 1 hr
at 4°C (positive selection). Five washes were then conducted, discarding
the supernatant from each wash. Cells were then scraped from the plate,
and washed twice with 15 mL of PBSCMA to collect the mammalian and yeast
cells. The cells were then spun down at 3,500×g for 10 min, resuspended
in SD-CAA, and grown overnight. The following day, yeast were induced in
SG-CAA and incubated for 2 days.
Subsequent rounds were carried out identically to round 1 with the
following exceptions: (i) selections were carried out in a single well
of a 6-well plate; (ii) 1 mL of PBSCMA was used for each wash; and (iii)
108 yeast cells and 1.2×106mammalian cells (83:1 ratio) were co-incubated.