Rapid fluorometric quantification of monocyte attachment in tissue culture wells.
A simple fluorometric assay that permits rapid quantification of attachment of monocytes or macrophages in tissue culture wells is described. Using 4,6-diamidino-2-phenylindole (DAPI) as a specific fluorochrome marker for DNA, we observed a dose-dependent increase with strong linear correlation in fluorescent emission over a broad range of DNA concentrations. Measurements of the DNA content of the human monocytic cell line THP-1 demonstrated a linear correlation between fluorescence intensity and cell number from 5 x 10(4) to 1 x 10(6) cells, with an estimated average DNA content of 7.5 pg DNA per cell. While untreated THP-1 cells were not detectably adherent, PMA induction for 24 h results in 57-76% adherence to plastic surface. This method was found to be useful for measuring the number of peripheral blood monocytes separated from lymphocytes by attachment. 16 subjects were sampled and the standard deviation of each individual did not exceed 10%. The number of attached cells was between 10-16% of the total mononuclear cells. Fluorescence measurement of DNA with DAPI permits rapid and accurate determination of cell numbers and appears useful in the quantification of adherent populations such as myelocytic cells and cell lines.
Shapira
L
L
Department of Periodontology, Eastman Dental Center, Rochester, NY 14620.
Takashiba
S
S
Kalmar
J R
JR
Van Dyke
T E
TE
Barak
V
V
Soskolne
W A
WA
D002448:Cell Adhesion; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D046508:Culture Techniques; D004247:DNA / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D007211:Indoles / Q000032:analysis; D007958:Leukocyte Count; D009000:Monocytes / Q000378:metabolism*; D010969:Plastics
D005456:Fluorescent Dyes; D007211:Indoles; D010969:Plastics; C007293:DAPI; D004247:DNA