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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-α and IL-1β, measured by ELISA. Results The purification procedure results in non-active but stimulation-competent monocytes with high yields (2.3–9 × 107 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. [ABSTRACT FROM AUTHOR]