A fluorometric deoxyribonucleic acid assay for tridimensional lattice cultures of fibroblasts.
A fast and sensitive in situ assay of deoxyribonucleic acid in miniaturized lattice cultures of fibroblasts is described. Tridimensional collagen and fibrin lattices prepared in 24-well plates were seeded with 50,000 to 200,000 cells. Cultures were fixed with formaldehyde, rinced with isopropanol, and dried. DNA assay was performed directly in the wells by addition of 3,5-diaminobenzoic acid (DABA) reagent. A calibration curve was prepared with calf thymus DNA. Fluorescence of DNA-DABA was evaluated after 45 min incubation (excitation wavelength 420 nm, emission wavelength 490 nm). The method showed linear results from 0.5 to 10 micrograms DNA and proved sensitive for low cell numbers (50,000 per dish). DNA assay in monolayers and in different types of lattices showed that comparable results were obtained in the different models without interference of the extracellular matrix. This technique is regarded as a costless and efficient tool for evaluating the number of cells in lattices in basal conditions or under pharmacological stimulation.
Laboratory of Biochemistry, CNRS ERS 0017, Faculty of Medicine, University of Reims-Champagne-Ardenne, France.
P
P
Gillery
A
A
Bonnet
J P
JP
Borel
D062365:Aminobenzoates; D000818:Animals; D002138:Calibration; D002417:Cattle; D002478:Cells, Cultured; D003094:Collagen; D004247:DNA / Q000032:analysis*; D005337:Fibrin; D005347:Fibroblasts / Q000737:chemistry* / Q000166:cytology; D005470:Fluorometry; D005557:Formaldehyde; D006801:Humans; D012680:Sensitivity and Specificity
D062365:Aminobenzoates; C017856:3,5-diaminobenzoic acid; D005557:Formaldehyde; D005337:Fibrin; D003094:Collagen; D004247:DNA