Isolation of non-activated monocytes from human umbilical cord blood.
OBJECTIVE
Methods for monocyte purification are common but few work with umbilical cord monocytes that do not activate the cell for subsequent culture analysis.


METHODS
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%).


CONCLUSIONS
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.
Normann
Erik
E
Department of Women's and Children's Health, Uppsala University Children's Hospital, Uppsala, Sweden. erik.normann@kbh.uu.se
Lacaze-Masmonteil
Thierry
T
Winkler-Lowen
Bonnie
B
Guilbert
Larry J
LJ
D004797:Enzyme-Linked Immunosorbent Assay; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D009000:Monocytes / Q000166:cytology*; D013048:Specimen Handling / Q000379:methods