2.9 Suspension cell-based enrichment via MACS
Non-transduced CHO-K1 (PD-L1- CHO-K1) cells and PD-L1-transduced CHO-K1 (PD-L1+ CHO-K1) cells were grown to 70-90% confluency, detached with trypsin-EDTA, and quenched via addition of culture medium. Cells were pelleted at 400×g for 5 min, washed three times with PBS pH 8, and then biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific). Per manufacturer’s protocol, PD-L1- CHO-K1 and PD-L1+CHO-K1 cells were each resuspended at 25×106 cells/mL in PBS pH 8 containing 2 mM of EZ-Link Sulfo-NHS-SS-Biotin reagent and incubated at room temperature for 30 min with rotation. Three washes were then conducted using PBSA (pH 7.3) to quench the reaction and remove excess byproducts. Mock library yeast induced in SG-CAA were pelleted at 3,500×g for 5 min, washed twice with PBSA, and resuspended in 5 mL of PBSA containing biotinylated PD-L1- CHO-K1 cells. In round 1, 107 PD-L1- CHO-K1 cells were incubated with 10-fold diversity of the yeast mock library (1010 yeast) for a final yeast:mammalian cell ratio of 1000:1. The cell mixture was then incubated for 1 hr at 4°C with rotation (negative selection). After 1 hr, 250 µL of magnetic beads coated with SA (Miltenyi Biotec) were added to the cell mixture and incubation proceeded for 20 min at 4°C with rotation. The cell mixture was then washed once with PBSA and centrifuged at 400×g for 5 min. The pellet was gently resuspended in 20 mL of PBSA and cells were separated over four magnetic columns (Miltenyi Biotec) (≈5 mL/column), according to the manufacturer’s protocol. The flow-through solutions from each column were pooled and pelleted at 3,500×g for 5 min. The pellet was then resuspended in 5 mL of PBSA containing biotinylated PD-L1+ CHO-K1 cells (107PD-L1+ CHO-K1 cells were used in round 1) and allowed to incubate for 2 hr at 4°C with rotation (positive selection). After 2 hr, 250 µL of magnetic beads were added to the cell mixture and incubation proceeded for 20 min at 4°C with rotation. The cell mixture was then washed once with PBSA and centrifuged at 400×g for 5 min. The pellet was gently resuspended in 20 mL of PBSA and separated over four magnetic columns (≈5 mL/column), according to the manufacturer’s protocol. Eluted cells from each column were pooled and centrifuged at 3,500×g for 10 min. The pellet was then resuspended in SD-CAA and grown overnight. The following day, the yeast were induced with SG-CAA and incubated for 2 days.
Subsequent rounds were carried out identically to round 1 with the following exceptions: (i) 108 yeast cells and 106 mammalian cells (100:1 ratio) were co-incubated; (ii) 50 µL of magnetic beads were incubated with the cell mixture; and (iii) 1 magnetic column was used for separation of the yeast/mammalian cell mixture.