Recombinant human serum albumin

Aqueous phase I contained either the isolated cells (cellular measurements) or purified monoclonal antibodies (calibration experiments). For the list of calibration antibodies used, please refer to Supplementary Table 1. The isolated cells were centrifuged at 300 × g for 5 min, then the cell pellet was gently re-suspended in cell medium to achieve a concentration of 5–10 million cells/ml. In order to minimize antibody secretion before droplet formation, the cell suspension was prepared just before droplet formation; and kept on ice for short-term storage. Cell medium was composed of RPMI 1640 with no phenol red, supplemented with 0.1% Pluronic F-68, 25 mM HEPES pH 7.4, 10% KnockOut Serum Replacement (all ThermoFisher), and 0.5% recombinant human serum albumin (Sigma Aldrich, A0237).

Bulk cell sorting and single‐cell deposition cloning was performed using a BD Influx cell sorter (BD Biosciences) based on a method described previously (Evans et al., 2015). For the bulk sort, 2 × 10⁷ cells were treated with nnAA for 2 or 4 hr and harvested by centrifugation. The cells were washed and stained with AF488‐conjugated anti‐human IgG (Fc specific) using sorting buffer containing PBS, 0.5% recombinant human serum albumin (Sigma, St. Louis, MO), 5 mM ethylenediaminetetraacetic acid (Life Technologies) and 25 mM HEPES (Calbiochem, San Diego, CA). Based on high and low AF488‐fluorescence intensity gated fractions, 2.5 × 10⁵ cells were deposited into 5‐ml collection tubes containing the culture medium. The sorted cells were centrifuged, resuspended in 2.5 ml fresh culture medium and plated into six‐well plates. For single‐cell cloning, 1 × 10⁶ cells (nnAA‐treated and stained with AF488‐conjugated anti‐human IgG [Fc specific] antibody) were sorted from the AF488‐gated fraction by depositing one cell per well into individual wells of 384‐well plates containing conditioned medium. All plates were incubated at 37°C in a humidified atmosphere with 6% CO₂ for outgrowth.