Clonacell hy medium c

The selected mice were anesthetized, and splenectomy was performed using an aseptic technique. Splenocytes were filtered through a 100 µm cell strainer (Falcon, 352360) in a sterile 50 mL conical tube, followed by washing 3 times with serum-free medium (360×g at 25°C for 7 min). Splenocytes and myeloma cells were counted using a Countess 3 FL automated cell counter (Invitrogen). Then, splenocytes and myeloma cells were mixed at a ratio of 2:1. The cell mixture was washed 3 times with 10 ml sterile DPBS buffer (Gibco, 14040–133), followed by 3 times 10 ml sterile BTX fusion buffer. The cell mixture was dissolved in ice-cold BTX fusion buffer (5×10⁷ cells/ml) and transferred into electrocuvettes (400 µl/cuvette). Electrofusion was performed using the BTX ECM 2001 machine. Fused cells were recovered in ClonaCell-HY medium C (STEMCELL, 03803) at 37°C in a 5% CO₂ humidified incubator overnight.

A semisolid medium containing HAT (hypoxanthine, aminopterin, and thymidine) was used for hybridoma selection and cloning. Briefly, recovered cells were harvested (360×g at 25 °C for 7 min) and reconstituted (2×10⁷ cells/ml) in ClonaCell-HY medium C (STEMCELL, 03803). Cell solutions were then gently mixed with semisolid ClonaCell-HY medium D (STEMCELL, 03804) at a ratio of 1:9 (v/v) according to the ClonaCell-HY technical manual instructions. The cell mixture was plated (2.5 ml/well) into a 6-well cell culture plate (Nunc, 140,675) and incubated at 37°C in a 5% CO₂ humidified incubator for 7–10 days. Four to six hybridoma colonies were randomly picked by pipetting and transferred to each well (mini-pool) of a 96-well culture plate containing 200 µl of ClonaCell-HY medium E (STEMCELL, 03,805). Mini-pools were cultured for 2 days, and the culture supernatants were taken to determine the hPD-1-specific IgG levels by ELISA.