Migration of mononuclear cells in the modified Boyden chamber as evaluated by DNA quantification and flow cytometry.
In vitro migration of mononuclear cells in the modified Boyden chamber was evaluated using flow cytometry and DNA quantification (Hoechst 33258) of all adherent and nonadherent cells. The effects of different membrane pore sizes, cell concentrations and incubation times were studied. Pore sizes of 3 and 5 microm resulted in a reduction in the number of nonadherent cells compared with a pore size of 8 microm. Reducing the incubation time from 60 to 40 and 20 min resulted in too few migrating monocytes for analysis by flow cytometry. Flow cytometry showed that both monocytes and lymphocytes migrated and adhered to the membrane when using peripheral blood mononuclear cells (PBMC) to study monocyte migration. Migration of lymphocytes under these conditions is a novel observation. A substantial number of migrated cells could be identified by flow cytometry and quantified by DNA measurement as nonadherent below the membrane. Samples of synovial fluid (n = 49) and plasma (n = 133) as chemoattractants analysed in triplicate resulted in mean coefficient of variation (CV) values of 11 and 9%, respectively. Variation from assay to assay on the same day, using N-formyl-methionyl-leucyl-phenylanine (fMLP) 10(-7) M as chemoattractant resulted in a CV of 13%. Day-to-day variation, using fMLP 10(-7) M as chemoattractant and the same well on three different days, resulted in a CV of 21%. These results were obtained using a pore size of 5 microm, a PBMC concentration of 3 x 10(6)/ml and 60 min of incubation. The combination of DNA quantification and flow cytometry thus allowed characterization and quantification of subsets of migrating adherent as well as nonadherent mononuclear cells.
Ellingsen
T
T
Department of Rheumatology, Arhus University Hospital, Denmark.
Storgaard
M
M
Møller
B K
BK
Buus
A
A
Andersen
P L
PL
Obel
N
N
Stengaard-Pedersen
K
K
D015703:Antigens, CD / Q000032:analysis; D001790:Blood Physiological Phenomena; D002630:Chemotactic Factors / Q000494:pharmacology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D003584:Cytological Techniques / Q000295:instrumentation*; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry; D006801:Humans; D007958:Leukocyte Count; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000166:cytology*; D008567:Membranes, Artificial; D009000:Monocytes / Q000737:chemistry / Q000166:cytology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D013582:Synovial Fluid / Q000502:physiology; D013601:T-Lymphocytes / Q000737:chemistry / Q000166:cytology; D013997:Time Factors
D015703:Antigens, CD; D002630:Chemotactic Factors; D008567:Membranes, Artificial; D009240:N-Formylmethionine Leucyl-Phenylalanine; D004247:DNA