Hi Julieta,

I have work with THP1 macrophages (obtained by PMA differentiation), I study ROS induction by different bacterial strains using this model.

Always, I use a positive control of infection (THP1 macrophages differentiated with 20nm PMA) and nonstimulated cells (noninfected PMA treated cells as a negative control and considered as a baseline level for ROS). In my experiments, I add the NBT solution in the media without any wash, following the next protocol:

150,000 macrophages were plated in each well in 96 well plate, then infect with different bacterial strains, at any time of infection 30 µl 0.1% Nitro blue tetrazolium chloride solution were added and then incubate for 15 min at 37ºC, finally the reaction was stopped by adding 1% SDS/0.08N NaOH ( overnight incubation for formazan solubilization, without DMSO or Formamide utilization), supernatants were transferred to a new plate to read at 570nM.

I hope this comment will improve your experiments, or you can give me more information about your complications.

Oh, I have not  before about using SDS, thanks for the help!

hi,

i was using NBT for detection of ROS in infected THP-1 cells infected with M. avium and followed this protocol:

Infection of cells were carried out  in 96 wells cell culture plates at MOI of 10:1.

At each time point, the supernatants were discarded and the cells

were incubated with 50ul of a 1 mg/ml NBT solution made in PBS.

After 45 min the reaction was stopped with 100 ul of 1 N

hydrochloric acid and the plates were shaken by using a shaker

(Heathrow Scientific Sea Star version 1.11) at 60 rpm for 10 min at

20°C. The wells were then washed with PBS at 37°C and shaken

again for 10 min at 20°C. The washed cells were then re-suspended

in 150 µl of DMSO (Sigma, USA) and 10ul of 1N sodium hydroxide

was added to generate the colour. The optical densities of the

reactions were determined by measuring the absorbance at 630 nm

using a micro plate reader (Microplate absorbance reader, LT-4000,

Labtech, Sussex, and UK). Positive controls of  THP-1 cells

were treated with 10 ul of LPS at 50 µg/ml as a final concentration

and negative controls wells of mock-infected (PBS).

hopefully, this protocol will help you.