I have LPS (powder) at -20,but I'm planing to dissolve it and use it to induce cell activation (macrophage/fibroblast). How should I divide the stock solution? Can I keep those solutions at -20 and for how long?
In the past I made 5-10 µL aliquots of 5 mg/mL LPS in PBS and stored it at -20C. They were good for almost 2 years. Lately, I use a ready-made solution in a glass bottle from Sigma of 1 mg/mL in aqueous solution (I think just ddH2O?) which is stored at 4C. So, I think there's a lot of flexibility. I think the only requirement is that if you are storing it in plastic, it should be at least 5 mg/mL to avoid major effects of LPS adsorption onto plastic. Storage in glass is preferred.
LPS is usually recommended to be first dissolved in endotoxin-free water (Yes, as suggested by Dr. Yaron, at high concentrations like 5 mg/mL; or 1 mg/mL as the next dilution). Store portions that you plan to use soon in small aliquots (20-30 uL) in 0.6mL tubes. Keep frozen at -20 or -80C. (flash freeze the aliquots in liquid nitrogen).
Once you take out one aliquot for use, keep it at 4C (when not finished up in experiments), and avoid refreezing.
LPS should not be diluted to below 100 ug/mL for storage in liquid form. It will gradually stick to the containers' walls. Avoid storing it as liquid at 4C for weeks before use.
When frozen and in mg/mL range, it's generally safe and stable.
Please search for product data sheet. Usually all information is given there.
Almost all above comments are OK.
Because of various reasons, we have been taking the amount of powder of LPS needed for a week or two, dissolved it in the aliquots each needed for one day use only (in appropriately small eppendorf cryo tubes), and kept them all on -20°C. Each lot of LPS has its specific characteristics ("power") so you must use the same lot for the controls and the experimental groups. To disolve, use the solution that you will use in your experiments.
Again I was reading a lot of papers and I'm not sure what volume of LPS should be added per well? I see that others have used cocn from 0.1ug-1ug/ml but no one is telling what volume is added per well. Because if you add 100ul of medium and another 100ul of test substance there is not much left for LPS...or does that means that I can pick what ever volume of LPS I want?
I am not sure I understand you. Make the smallest possible but highly concentrated volumes of the aliquts of LPS. You can then easily arrive at the final concentration needed even in small volumes of the solution and have final solution as you want. If you would disolve LPS in the same solution that you use as your medium, all what you will have to care will be the final concentration of LPS.
I was not talking about stock solution, I was talking about working solution that is to be added to plate.
In the surroundings of the cells you need to have a given concentration, this is all. The question of volume is another question that depends on the other experimental conditions also. Please explain exactly what is this you are doing.
Well I will try to evaluate the influence of different plant extracts on LPS stimulated macrophages through their production of NO
May I ask, how about the preparation of LPS solutions to induce into the embryos of zebrafish? is the preparations still the same? or should i dilute the stock solutions (LPS+PBS) with embryo media afterwards? Any helps would be very much helpful and thank you in advance!
Having some troubles with LPS again. Even-though I made fresh solution from powder (Sigma) there is no significant stimulation of NO production in primary macrophages. Few week before old solution worked fine, and one day it just stoped working. I decided to make a fresh stock solution from a same LPS and there is no reaction as well. Can anyone think of a reason?
Nikola M Stojanovic I have the same problem as yours, suddenly my LPS stopped its stimulation effect on my cells and I do not know why!!! I have you found a solution?
You may inject to a mouse a solution of LPS 0.5ml (5mg/kg, in 0.9%NaCl) i.p. It will die after 20 hours, This my be a test for the efficacy of LPS.
Can I dissolve LPS in normal saline to a concentration of 5mg/ml? I just want to be sure before I start
Hi Kisipan, you can make 5mg/mL LPS solution in PBS. Please see following reference:
https://media.cellsignal.com/pdf/14011.pdf
So, I guess normal saline should also work. Good luck!
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