Saturated ammonium sulfate solution

The LC-4 hybridoma culture supernatant was collected every 3–4 days depending on the density of the cells. Proteins in the culture supernatant were precipitated by the addition of saturated ammonium sulfate solution (Thermo Fisher Scientific) until the solution became turbid. The solution was centrifuged at 3000× g for 30 min at 4 °C. The pellet was resuspended in a small volume of phosphate buffer saline (PBS) and dialyzed against PBS overnight at 4 °C using Slide-A-Lyzer 10K dialysis cassettes (Thermo Fisher Scientific). The resulting antibody was measured for concentration using a Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific) and 2 μL and 10 μL of purified mAb was further analyzed for purity by Coomassie staining and Western blot. For the latter, the blot was probed with 1:2000 of goat anti-mouse IgG horseradish peroxidase (HRP), washed, and visualized using the Pierce^TM ECL Western Blotting Substrate (Thermo Fisher Scientific).

The purification and characterization of anti-chicken CD4 and CD8 mAbs were performed as described by Umthong et al. [42 (link)]. The supernatants from cell cultures of Lc-6 and Lc-4 were collected every 3–4 days and proteins were precipitated by saturated ammonium sulfate solution (Thermo Fisher Scientific). The supernatants were centrifuged, and the pellets were resuspended in 5–8 milliliters of PBS, depending on the size of the pellets. The protein suspensions were dialyzed against PBS overnight at 4 °C in dialysis cassettes (Slide-A-Lyzer, Thermo Fisher Scientific). The modified route of delivery and concentrations of mAbs needed for the efficient depletion of CD4⁺ and CD8⁺ T cells were based on the study by Umthong et al. [42 (link)].