A tetrazolium-based colorimetric assay for quantification of HIV-1-induced cytopathogenicity in monocyte-macrophages exposed to macrophage-colony-stimulating factor.
A sensitive assay was developed for in vitro evaluation of anti-HIV agents in monocyte-macrophage cells (M/M) (a crucial target of HIV in the body). Monocyte-macrophage cells are usually poorly sensitive to the cytopathic effect induced by HIV. However, when fresh adherent monocyte-macrophage cells are cultured at relatively high density in the presence of macrophage-colony stimulating factor (M-CSF), they undergo cytolysis and die in 2-3 weeks. HIV-mediated cell-killing can thus be assessed with a method based on the reduction of the yellow colored 3-(4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by metabolically active cells to a blue formazan, which can be measured spectrophotometrically. HIV-mediated cytopathic effect of M-CSF-exposed monocyte-macrophage cells was consistently achieved in all experiments performed under the conditions described herein. Anti-HIV activity of zidovudine (AZT) was also comparatively evaluated in M-CSF- and normal monocyte-macrophage cells both using the MTT assay and by measuring HIV-p24 antigen production in supernatants of monocyte-macrophage cells cultures, and similar results obtained with both methods. These results support the use of this colorimetric assay for broad screening of anti-HIV agents in monocyte-macrophage cells.
Bergamini
A
A
Department of Public Health, University of Rome, Tor Vergata, Italy.
Perno
C F
CF
Capozzi
M
M
Mannella
E
E
Salanitro
A
A
Caliò
R
R
Rocchi
G
G
D003124:Colorimetry / Q000379:methods; D004396:Coloring Agents; D003588:Cytopathogenic Effect, Viral; D005069:Evaluation Studies as Topic; D015497:HIV-1 / Q000187:drug effects* / Q000472:pathogenicity; D006801:Humans; D066298:In Vitro Techniques; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000382:microbiology; D008826:Microbial Sensitivity Tests / Q000379:methods; D009000:Monocytes / Q000187:drug effects / Q000382:microbiology; D013778:Tetrazolium Salts; D013844:Thiazoles; D014773:Virology / Q000379:methods*; D015215:Zidovudine / Q000494:pharmacology
D004396:Coloring Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D015215:Zidovudine; D016173:Macrophage Colony-Stimulating Factor; C022616:thiazolyl blue