Isolation of non-activated monocytes from human umbilical cord blood.

PROBLEM: Methods for monocyte purification are common but few work with umbilical cord monocytes that do not activate the cell for subsequent culture analysis. METHODS OF STUDY: The collection procedure avoids use of needles and procedures that variably activate blood clotting and uses a purification procedure that involves diluted Ficoll, autologous serum to remove platelets and 42% and 51% Percoll step gradients for the final purification. The resulting monocytes were stimulated with bacterial lipopolysaccharide and formalin-treated bacteria Escherichia coli and group B streptococci (GBS) to secrete TNF-alpha and IL-1beta, measured by ELISA. RESULTS: The purification procedure results in non-active but stimulation-competent monocytes with high yields (2.3-9 x 10(7) cells) and purity (from 70% to 98%). CONCLUSION: We describe a procedure that is easy, uses common reagents and provides a uniformly high yield and purity of non-activated fetal monocytes for studies of innate defense responses.