5 replies to this topic

[Crazycell]

Hi,everyone,

I have several natural products and want to evaluate them the activity of NO production inhibition

My experiment:

cell: RAW264.7  1*105 per well

culture: H-DMEM (10% FBS) without phenol red

LPS concentration: 10, 1, 0.1 ug/mL (sigma) dissove in PBS and add to the well

Detection of NO2- : Griess reaction (promega)

The Problem is the NO amount of the LPS treated well was very low (about 10-20uM)

How to do?

[HomeBrew]

It looks like you had three LPS-treated wells.  What was the NO production in all of them?  Was there a dose response?

How much NO do you expect to be produced?

[almost a doctor]

Crazycell, on Jul 2 2009, 07:32 AM, said:

Hi,everyone,

I have several natural products and want to evaluate them the activity of NO production inhibition

My experiment:

cell: RAW264.7  1*105 per well

culture: H-DMEM (10% FBS) without phenol red

LPS concentration: 10, 1, 0.1 ug/mL (sigma) dissove in PBS and add to the well

Detection of NO2- : Griess reaction (promega)

The Problem is the NO amount of the LPS treated well was very low (about 10-20uM)

How to do?

Macrophages dont make much NO in response to LPS unless you add IFNgamma at the same time.
100ng/ml LPS + 10U/ml INFg will give you NO concentrations of ~50uM

Hope this helps.

[Crazycell]

Thank you very much for the suggestion! And I will try it again with IFN-gamma.

almost a doctor, on Jul 2 2009, 06:12 AM, said:

Crazycell, on Jul 2 2009, 07:32 AM, said:

Hi,everyone,

I have several natural products and want to evaluate them the activity of NO production inhibition

My experiment:

cell: RAW264.7  1*105 per well

culture: H-DMEM (10% FBS) without phenol red

LPS concentration: 10, 1, 0.1 ug/mL (sigma) dissove in PBS and add to the well

Detection of NO2- : Griess reaction (promega)

The Problem is the NO amount of the LPS treated well was very low (about 10-20uM)

How to do?

Macrophages dont make much NO in response to LPS unless you add IFNgamma at the same time.
100ng/ml LPS + 10U/ml INFg will give you NO concentrations of ~50uM

Hope this helps.

[Crazycell]

NO concentration that I want is 50 uM, which is reported by many others.

HomeBrew, on Jul 2 2009, 05:24 AM, said:

It looks like you had three LPS-treated wells.  What was the NO production in all of them?  Was there a dose response?

How much NO do you expect to be produced?

[warsel]

measuring NO as NO2- you need to be careful:
1) some compounds (like reducing agents) can release NO from NO2- meaning your results may be unpredictable or lead you to draw wrong conclusions.
2) NO2- is not the only "storage form" of NO, you may want to consider NO3 as well.

this article might be of interest to you