[Improved MTT colorimetric assay for serum TNF activity]

Area of Science:

• Biochemistry
• Immunology
• Cell Biology

Background:

• Serum tumor necrosis factor (TNF) activity is implicated in the pathogenesis and prognosis of numerous diseases.
• Existing methods for assaying serum TNF activity lack economic viability and reliability.
• A need exists for an improved, cost-effective, and dependable assay for serum TNF.

Purpose of the Study:

• To develop an economical and reliable method for assaying serum TNF activity.
• To establish an improved 3-(4,5-dimethyl thiazoly)2,5-diphenyl-tetrazolium bromide (MTT) colorimetric assay.
• To validate the improved MTT assay for measuring TNF activity in healthy individuals' peripheral blood serum.

Main Methods:

• An improved MTT colorimetric assay was established using a neutrophil solvent (20% SDS-50% DMF) instead of acidified isopropanol.
• This method measures the amount and function of living cells by quantifying the purple formazan metabolite.
• The assay was applied to measure TNF activity in the peripheral blood serum of 20 healthy persons.

Main Results:

• The improved MTT assay demonstrated a positive correlation between living cell number and optical density (OD570nm-OD630nm) (r=0.87, P<0.01) when target cell activity was ≥95% and density was ≥1x10^2/well.
• A negative correlation was observed between TNF level and OD volume (r=0.79, P<0.01) at a target cell density of 5x10^4/well.
• The mean TNF activity in 20 healthy individuals was 20.95 ± 3.2 IU/ml, with good repeatability within 3-12 hours.

Conclusions:

• The improved MTT colorimetric assay is a dependable, easy-to-perform, and economical method for measuring serum TNF activity.
• This assay avoids protein deposition issues seen in conventional MTT assays, offering enhanced repeatability.
• The method provides a reliable means to assess serum TNF levels in healthy populations.