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.