Monocyte isolation by flow cytometer-monitored centrifugal elutriation: a preparative tool for antibody-dependent cell-mediated cytotoxicity (ADCC) assays.
The value of isolating monocytes by flow cytometer-monitored centrifugal elutriation (CE) after initial density gradient centrifugation (DGC) was investigated. A pooled cell suspension prepared by DGC+CE had a monocyte purity of 90% while a pool prepared by DGC alone had a monocyte purity of only 30%. Monocyte suspensions prepared from 15 separate blood donations demonstrated that, despite wide variation in initial monocyte purity following DGC (i.e. 13-32%), CE consistently increased the purity to 85-92%. Antibody-dependent cell-mediated cytotoxicity (ADCC) studies showed that the most sensitive and reproducible assays were those performed with the DGC+CE pool. At a monocyte concentration of 2 x 10(6)/ml the DGC+CE pool was almost twice as active as the DGC pool (60.2% lysis vs 31.2% lysis, respectively). Considerable variation in ADCC activity was seen when using monocytes from individual donors. We conclude that centrifugal elutriation is a valuable tool for preparing effector cells for use in monocyte driven ADCC assays.
North London Blood Transfusion Centre, Colindale, England, UK.
S F
SF
Garner
A R
AR
Thomson
A
A
Lubenko
J
J
Savage
D000920:Antibody-Dependent Cell Cytotoxicity; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D005434:Flow Cytometry; D006801:Humans; D009000:Monocytes / Q000276:immunology*