Hello,
I am hoping you can assist me (or direct me to someone who can). I am trying to establish a lethal dose of LPS 055:B5 (Sigma, phenol extraction). We are using D-Galn (Sigma, hydrochloride) at 800mg/kg and I have used LPS doses ranging from .2mg/kg to 80mg/kg, and I have not produced any lethality. We are using outbred ICR males (5-8 week olds), IV tail injections and reconstituting the D-Galn and LPS (we make fresh LPS each time 20mg/ml in PBS then take the concentration down from there) in saline and heating in a 37C shaker bath for 30 minutes prior to injecting. Any insight you could provide will be greatly appreciated.
I havn't tried sub-Q (I have never heard them referenced in any of the literature) although I am familiar with them. I considered IP, alot of the literature uses IP as opposed to IV, yet to me IP seems less accurate - I can easily tell whether I am in the vein or not, yet other than seeing anything other than air in the hub of the syringe when aspirating I can not be sure whether I am in the peritoneal cavity or not. I am very comfortable with IV and since I have seen them used in the literature it is not a variable I have yet considered changing. I recently purchased a batch of 0111:B4 LPS and I tried it on four groups (10ug/mouse with 20mg D-galactosamine and 10ug/mouse with 40mg D-galn and then 100ug/mouse with each differing D-galn dose) and this time instead of vortexing I sonicated (for approx. 1 min) - same results - sick mice but not dead mice. This week I will be ordering an inbred strain C3H/HeN- I will be shocked yet pleased if this is what makes the difference- any other ideas?
I have never tried retro orbital - I am definately hitting the vein in the IV tail injections(worked part time as a vet tech when I was in school) and I have used LPS doses as high as 1600ug/mouse with the galactosamine sensitization - I hesitate to go higher mostrly due to literature saying that they have acheived lethality with as low as 10ug/mouse. What does MTD stand for? I have had other researcher's comment that inbred mice and particularly females are somewhat more susceptible to LPS so....the mice are arriving tomorrow - I'll keep posting - hopefully with better results
I have finally seen some lethality - the variable I changed to get it was the mice
using C3H females and 24 hours in I have 75% death in 2 groups - .7mg/kg LPS and 1.2mg/kg LPS both sensitized w/D-Galactosamine - I will be repeating the same today
Next I will lower the dose to find the lowest dose to produce 100% lethality (within 48 hours) per group (n=4). My company works with hyper immunized horses - we pherese them and sell the plasma to other horse owner for the Ab's - so after I find the lowest lethal dose I will challenge the mice with that dose and hyper immunized plasma to determine the efficacy of our (hopefully) product.
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