HELP! LPS stimulated plasma gone awry

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Nimoy
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HELP! LPS stimulated plasma gone awry

I have cultured rat blood and stimulated it with LPS... I followed a protocol I found in the literature, however, the levels of cytokines produced are at the lower limit of detection with my R+D systems TNF ELISA kit.... The levels in published are at least 20 times what I am finding.... Can anyone tell me how my cultures have gone wrong?

RockyDoc
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Nimoy,

Nimoy,

Did you find out what happened with your LPS-stimulated cells? I am currently working with other stimulants (Concanavalin A, PMA/IoM) and I am not getting the results reported in the literature. Just wondering whether I may be going through what you experienced?

Kind regards,
RockyDoc

Nimoy wrote:

I have cultured rat blood and stimulated it with LPS... I followed a protocol I found in the literature, however, the levels of cytokines produced are at the lower limit of detection with my R+D systems TNF ELISA kit.... The levels in published are at least 20 times what I am finding.... Can anyone tell me how my cultures have gone wrong?
Nimoy
Nimoy's picture
RockyDoc wrote:Nimoy,

RockyDoc wrote:

Nimoy,

Did you find out what happened with your LPS-stimulated cells? I am currently working with other stimulants (Concanavalin A, PMA/IoM) and I am not getting the results reported in the literature. Just wondering whether I may be going through what you experienced?

Kind regards,
RockyDoc

Nimoy wrote:

I have cultured rat blood and stimulated it with LPS... I followed a protocol I found in the literature, however, the levels of cytokines produced are at the lower limit of detection with my R+D systems TNF ELISA kit.... The levels in published are at least 20 times what I am finding.... Can anyone tell me how my cultures have gone wrong?
Nimoy
Nimoy's picture
Yeah, I did find out what the

Yeah, I did find out what the problem was.. I had used an EDTA coated tube, which was apparently suppressing my stimulated cytokine responses... I have now switched to sodium heparin coated tubes and the levels are much higher... Hope this helps!

Nimoy wrote:

RockyDoc wrote:
Nimoy,

Did you find out what happened with your LPS-stimulated cells? I am currently working with other stimulants (Concanavalin A, PMA/IoM) and I am not getting the results reported in the literature. Just wondering whether I may be going through what you experienced?

Kind regards,
RockyDoc

Nimoy wrote:

I have cultured rat blood and stimulated it with LPS... I followed a protocol I found in the literature, however, the levels of cytokines produced are at the lower limit of detection with my R+D systems TNF ELISA kit.... The levels in published are at least 20 times what I am finding.... Can anyone tell me how my cultures have gone wrong?
ycseattle
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Nimoy,

Nimoy,
Can you share the protocol for stimulating rat blood with LPS (eg. concentration LPS used vs TNF-a producted).
Thanks,
ycseattle
 
 

angelaevan
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I wondered if anyone has any

I wondered if anyone has any advice for my in vivo study - my protocol is awry and i don't know why

So far....
 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 0.2mg/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 37oC shaker bath for 30 minutes prior to injecting.  Any insight you could provide will be greatly appreciated.
 
I have just ordered LPS 0111:B4 and if it doesn't work on my 20 remaining ICR I am gonna try Balb-C's (inbred) but I only got 10mg so I need to figure this out fast.

newscientist2010
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I have been using O55:B5 LPS

I have been using O55:B5 LPS from Sigma.....it works perfectly fine. There is just a minor difference in mine and your protocol. First, I use distilled water to dissolve LPS instead of PBS. Second, I don't heat the solution prior to injecting (i.p.). I have not worked with ICR, but i have worked with Balb/c, you might consider checking out the basal TLR4 levels in ICR.

Good luck!!!