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Clonacell hy medium c

Manufactured by STEMCELL

ClonaCell-HY medium C is a specialized cell culture medium designed for the maintenance and expansion of human hybridoma cells. It provides the essential nutrients and growth factors required for the optimal growth and proliferation of these cells.

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2 protocols using clonacell hy medium c

1

Splenocyte-Myeloma Cell Fusion Protocol

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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.
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2

Hybridoma Selection and Cloning using HAT

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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.
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