Hi,
Hope you are doing well. I am doing my experiment to check cytokine profile response of LPS following U251 stimulation. At the beginning, I started to get the results then after I get the new lot of LPS (Sigma ), I am unable to stimulate my cells each time negative control and LPS stimulated cells yield the same cytokine profile. On the other hand when these cells are stimulated with poly I:C or JEV infection they yield the expected results. I have been trying again and again with LPS, but all in vain. What do you suggest.
I have also borrowed LPS from my neighbor laboratories (same type) but did not get any response. I am in need of your kind advise on this special aspect. Thanks for your time.
Ali Zohaib
DVM, Masters
Dear Ali,
LPS has different kinds, some of them are more antigenic than other depending on the species that extracted from it, Also LPS used for animal study is totally different from that for cell culture work. Even in sigma u will find a lot of kinds of LPS.
So try to select the best antigenic one, and try to read more literature so u can choose the best one.
Regards,
Bashir
This is a terrible age old problem. A cynical approach is to buy a large lot of the same LPS. Nobody else will be able to reproduce you results either!
Check the exact O glycosydation of the LPS. Try other vendors, SIgma is cheapest, but you get you pay for.
What cells are you using. Sometimes these mutate after years in culture and lose functional receptors; TLR4?
Hi,
You need to be more precise regarding the kind of LPS you are currently using or used in the past.
For example, LPS from pseudomonas aeruginosa is a really poor inducer.
In another hand LPS form E. Coli O111:B4 (form SIGMA) induces nice response.
ALexis
Hi Ali,
One thing I could suggest is to make sure ALL the reagents you are using with your cell culture are LPS/endotoxin free to remove any tolerogenic effects of these contaminants in your continuous cultures. Make sure you buy in any PBS in solution rather than make up your own from powder/tablets as homemade will always contain endotoxin even if autoclaved or otherwise sterilised.
Good luck.
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