Hi !
Anyone measured cytokine secretion (IL-1beta and TNF-alpha) from macrophages stimulated by K. pneumoniae?
I want to determine in cell culture of killing assay (30 minutes; MOI: 50)? Is time enough to quantify differences in pro inflamattory cytokine levels comparing to control?
Thank you
Hi,
Nope, at 30 min you can start detecting mRNA by real time or any other sensitive technique. For proteins to be secreted and be able to detect in supernatants by ELISA, you should wait at least 6h. Most people measure accumulation at 12 or 24h. In any case, if you have a very sensitive ELISA (or any other technique) for a given protein, and depending on your system, you can stretch your detection to as early as 2h.
Best
Fernando
Hi,
Nope, at 30 min you can start detecting mRNA by real time or any other sensitive technique. For proteins to be secreted and be able to detect in supernatants by ELISA, you should wait at least 6h. Most people measure accumulation at 12 or 24h. In any case, if you have a very sensitive ELISA (or any other technique) for a given protein, and depending on your system, you can stretch your detection to as early as 2h.
Best
Fernando
No. As Fernando mentioned, you may detect TNF message 30 minutes post infection. It is still too early for IL1B message. However, you may try to measure IL-18 release. This cytokine is constitutively expressed and readout doesn't depend on mRNA synthesis. 30 minutes is sufficient to prime the inflammasome. Add 5 mM of ATP for another 30 minutes (as signal two in two-step inflammasome activation model) and measure IL-18 release.
No. As both previous answers state you will need a significant accumulation of protein in supernatant for an ELISA to detect either TNF or IL-1. Depending on your cell and bacterial challenge you should see mRNA signal for CxC chemokines at around 15 mins post challenge and pro-inflammatory cytosine signals for 20-30 minutes post challenge. An mRNA signal will peak at around 1h for most of these. You should easily be able to use qRT-PCR to compare the relative expression between groups and there are plenty of protocols and off the shelf kits for this. I don't know much about Klebsiella but an MOI 50 seems a high challenge.
If you need a control for base level of secreted cytokines no harm if you takes supernatants after 30 min of killing. I wonder what kind of killing do you use? Any chance it can trigger the release of preexisting cytokines into sups?
Hi Natalie.
I use MTT assay.
I was wondering if I collect the medium of the cell culture before add LB broth. Is that possible to measure cytokines?
Because I know that macrophages stimulated by LPS release IL-6 and TNF-alpha in 30 minutes. So, I guess I will use only LPS as my positive control and RPMI as my negative control.
Well, if you are positive that LPS (presumably from Klebsiella?) does it, you can definitely do this. But if the levels are pitiful, like 20 - 50 pkg, it will be difficult to publish the data or see the difference between the groups. I wonder what question do you try to answer? I also wonder may be Elispot variant might be more convincing than ELISA in this case? Though again signal might be too low.
We have only ELISA system in our lab. When I use LPS, I saw differences between the groups in 30 minutes.
I have searching a paper to use as model, but I cant find one to reference.
What cytokines do you see in 30 minutes and what concentrations? Killing may result in nonspecific release of pro-cytokines which may be detected by ELISA.
At least TNFalpha is released promptly after a signal from the membrane. However, wash very well your microbas to leave out any cell debris from their medium. Killing of what? Microbas? Cells? Go through on this again.
Mikhail A Gavrilin
In 30 minutes we measured TNF-alpha and IL-6 released by resident peritoneal macrophages.
Attila Lehotzky
Before the challenge with LPS, the macrophages were washed tree times with warm RPMI.
Fernando, thank you for updating. This may be a difference because I was referring to human monocytes and macrophages. We don't see confident TNF and IL-6 release in 30 minutes ( but see it well for TNF in 1 hour). Mouse peritoneal macrophages may be different.
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