Quantifying Macrophage Metabolic Activity

THP-1 monocytes were seeded at 180 000 cells/well in 24 well plates and differentiated in macrophages as described. After incubation with IFN-γ ± LPS, cells were incubated 2 h with 500 μl of MTT reagent (2.5 mg/ml of PBS, Sigma #M2128) in the CO₂ incubator. The media were then removed and 1 ml of lysis buffer (SDS 30 %/N,N-dimethyl-formamide 2:1 pH 4.7) was added per well. Plates were incubated at 37 °C and gently shaked at 70 rpm for 1 h. The absorbance was then measured at 570 nm.

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Genin M., Clement F., Fattaccioli A., Raes M, & Michiels C. (2015). M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer, 15, 577.

Publication 2015

MTT reagent

Buffer Cells Dimethyl formamide Ifn γ Macrophages Monocytes

Corresponding Organization :

Other organizations : University of Namur

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Variable analysis

independent variables

IFN-γ

dependent variables

Cell viability (measured by MTT assay)

control variables

THP-1 monocytes seeded at 180,000 cells/well in 24-well plates
Differentiation of monocytes into macrophages
Incubation time with MTT reagent (2 h)
Lysis buffer composition (SDS 30%/N,N-dimethyl-formamide 2:1 pH 4.7)
Incubation time with lysis buffer (1 h)
Absorbance measurement at 570 nm

controls

Negative control: Cells incubated with no IFN-γ and no LPS

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