Hello, 

I am currently trying to standardise qPCR primers for IL-10 expression in human macrophages i.e. checking amplification, primer efficiencies. Before the actual stimulation by bacteria, I am using control treatments to establish the system using M-CSF mediated THP-1-derived macrophages. 

I am using the following treatments: 

1. Monocytes only

2. Differentiated but unpolarised Macrophages

3. LPS+ IFNg

4. TGFBeta1

5. Glucocorticoids mixture (GCM)

6. As a control for some experiments, PMA-mediated THP1 derived macros.

I have tested the following samples already for the expression of IL-10 and MMP12 with two separate sets of primers. 

1. Monos, 2. unpolarised macros 3. LPS + IFNg macros 4. GCM macros

Problems: 

1. I cannot detect IL-10  and MMP12 in monos (Amplified very late Ct 38 or undetermined).

2. Cannot detect IL-10 and MMP12 in M2-like (GCM treated) macros  (Amplified very late Ct 38 or undetermined).

3. Very weak MMP12 and almost no IL-10 in M1-like (LPS+IFNg) macros.

Suggestions:

1. Should I wait for the TGFbeta1 samples before concluding anything, is it usually expected in this treatment ?

2. Should I check in PMA-treated macros ? 

3. What could be a positive control for IL-10 and MMP12 ?

I would be extremely grateful if someone could show me the way here,

Cheers,

Sudip