Purification of human melanocytes by monoclonal antibody combined with Percoll TM gradients

Abstract

Cultures of human melanocytes obtained by differential plating of human epidermal cells in 12-myristate 13-acetate (PMA) were purified using a monoclonal antibody R 24 which detects a restricted glycolipid antigen present on melanoma cells and melanocytes. Melanocytes were rosetted using protein A-conjugated human red blood cells and separated from non-rosetted ftbroblasts on discontinuous Percoll ™ gradients. 100% pure cultures of melanocytes obtained in this fashion were then successfully grown in tissue culture in the presence of PMA and cholera toxin for at least thirty passages (corresponding to approx. sixty cell doublings). We conclude that: 1. 1. Pure cultures of melanocytes are a prerequisite for the establishment of long-term cultures. 2. 2. Since most human melanomas express substantial levels of R 21 antigen, this method can be applied to purification of melanomas and can be easily adapted to separation of subpopulations of melanocytes and melanoma cells recognized by specific monoclonal antibodies.