Control for no LPS in your stimuli - How to exclude the possibility you have LPS...... (Jul/25/2005 )

Hello Every one!

I need to stimulate PBMCs with a very higlhy cationic peptide and then read by QPCR the induction of IL-6, 8 and 10. If I did not perform the LAL test on my peptide's batch as I do not have enough peptide, how could I exclude results are from LPS contamination??? These peptides are very very resistant to heat inactivation and proteinase K alone induce IL-8

ANy help would be wonderful!


MIcky

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if you don't want to exclude the effect of LPS in your experiment simply try adding polymyxin B (a nice inhibitor of LPS) in a control well

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well, well old polymyxinB it is again, hm. you're right, it's a nice inhibitor of LPS, but was found to have some immunestimulatory properties itself (induction of cytokine secretion, maturation, apoptosis etc., and it's toxic, by the way) and should therfor be carefully monitored (i.e. use many well thought trough controls!).

But, besides the LAL assay (which is super senititve, but very easily led astray in the positive or negative direction), you could use LPS a positive contol, read out the cytokines it induces and find one that's differently induced by your peptide and LPS (i.e. go for the differences between LPS and your sample).

good luck

mike

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