GolgiPlug THP-1 Issue - Timecourse (Jul/28/2010 )
Hi All,
So I am doing a protocol using THP-1 cells. I stim with LPS and have been adding GolgiPlug after 2 hours giving the cells some time to make the cytokines I want to look at. But when I did a comparision with Flow Cytometry between adding GP right away with the LPS or wait 2 hrs, the results tell me that adding GP right after the LPS provides more positive cytokine readings than 2 hrs. I have looked around and others also recommend 2 hrs. I put in 1microL in 100 microL media to dilute, then add 1 mircoL of that to 50 microL of cells at about 10^6 per mL. LPS used at 100ng/mL (which has been working fine.) Thank you! Time sensitive!
Rudy
Hey, trust your results! If you get better staining by including the GolgiPlug from the start then by all means do it! I'm not all that surprised at your data. If you are looking at TNF, there's premade TNF in macrophages that is release upon stimulation really pretty quickly. So if you omit the GolgiPlug you will allow some of that out and decrease your staining. Other cytokines might be different but still, the point of the GolgiPlug (brefeldin A or whatever) is to prevent release of the cytokine into the medium. I would think that the sooner you add it the better.
rnazitto on Wed Jul 28 22:34:38 2010 said:
Hi All,
So I am doing a protocol using THP-1 cells. I stim with LPS and have been adding GolgiPlug after 2 hours giving the cells some time to make the cytokines I want to look at. But when I did a comparision with Flow Cytometry between adding GP right away with the LPS or wait 2 hrs, the results tell me that adding GP right after the LPS provides more positive cytokine readings than 2 hrs. I have looked around and others also recommend 2 hrs. I put in 1microL in 100 microL media to dilute, then add 1 mircoL of that to 50 microL of cells at about 10^6 per mL. LPS used at 100ng/mL (which has been working fine.) Thank you! Time sensitive!
Rudy