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.