Hello all, my current experiments involve using THP-1 cells that have been differentiated into immature dendritic cells. According to the literature, this is done by incubation of the cells for, from what I can tell, a minimum of 5 days in 100ng/mL GM-CSF and IL-4, replacing the media every 2 days.
Following this, we attempted stimulation of the cells with LPS (14.5umol/mL) and the results were poor. The cells did not respond the way an iDC should respond to LPS stimulation; we saw only a modest amount of TNFa after 48 hours and no IL-12p70.
I'm currently trying to trouble shoot the experiment. The cells were (and continue) to grow suitably, though at only half the rate at which ATCC says they will. The only thing I can think of that may have affected the differentiation was the handling of the cytokines; the GM-CSF and IL-4, after being diluted in media, were filtered through a 0.2um filter. I'd thought this wouldn't affect the cytokines, but perhaps I was wrong. Any thoughts?
For reference, here is the complete media we're using, not including the GM-CSF and IL-4:
1. RPMI 1640
2. 0.05mM β-2-Mercaptoethanol
3. 10% Fetal Bovine Serum
4. 1mM Sodium Pyruvate
5. 1X Pen-Strep (100IU/mL Pen, 100ug/mL Strep)
6. 2 mM L-Glutamine
Our freezing media is 90% FBS and 10% DMSO; we have ~85% viability after thawing.
HalMember Since 16 Sep 2009
Offline Last Active Jan 22 2013 10:04 AM
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Microbiology and Immunology