I tried to measure IL1β levels secreted by 1ug/mlLPS or 500ng/ml IFN-γ BV-2 cells and I didn't find any in the medium. My dilution was 20x but I'm worried that this cytokine is not cleaved (and therefore recognizable by the antibodies) due to ATP absence. Has anyone tried this with cell lines, and if successful, did you challenge the cells with ATP before the ELISA?
You do need to use ATP to activate inflammasome for most of of the cell line. As a matter of fact, I don't know any which additional ATP is not needed. IF you want to be sure whether IL1b is induced, you could do a western blot for cell lysate. The capture antibody in the ELISA works well for that purpose.
Good luck
You could check the system is working by spiking a sample and measuring ELISA. Or you could wait for a longer period before you measure the cytokine and do not dulite the sample.
Hi Theodore Kapellos,
You can prefer Cell lysate for estimating IL-1 beta has an alternate, add 100 µL of 0.1% Triton X 100 in DPBS in case if you use 96 well plate, this will be helpful to concentrate the protein in the lysate and freeze it at -80 o C. Before performing your ELISA you can freeze thaw the sample for few times (this may required optimization) for complete lysis of the cells. And don't dilute your sample until it is required. You need to optimize this part too, where in at first you have to add neat samples and observe the color development with respect of your Standard color development, if your sample signal reading fall beyond your standard signal / saturates then you have to dilute the sample. You can express the results in respect to total protein concentration, if you perform protein expression.
Do time response curve for IL-1 beta secretion, usually IL-1 beta will response quickly to inflammation, and lost for short time in medium. So you have to check your sample even at 4th hour. In case of macrophage IL-1 beta usually peaks between 7-9 hrs after induction with inflammatory substance
All the best
Unfortunately, cells were used for RNA extraction so I only have the S/Ns. I have done a time course of the secretion, but the problem is that my samples in the ELISA didnt work! I could definitely repeat with undiluted samples but I dont want to waste any sample until I know whether it is indeed detectable without addition of any other reagent in the S/N.
Hi Theodore Kapellos
Try to re do the experiment this time use cell lysate at least try for two range one controls another IC50 value of your Test compound in other estimation this may give you wide idea of where your protein is present.
You may increase your cell seeding density too in order to get better signal (again optimization is required), well volume can be reduced since it can too dilute the sample example if you add 2 ml to 24 well plate you can reduce the volume if your cell seeding density is less if you increase the density then respectively you have to increase the volume too.
Check for your kit specificity, detection range, Expiry date, even you can check for failure of the kit on specific batch (if your standard were working then this wont work out) still if they din't show proper signal window with in the different concentration also within incubation time referred by the manufacture then 1) kit may expire 2) batch failure.
You may have to look for Cell passage number, morphology, cytotoxicity (if your test compound is reducing the MTT or other dye then you may get false positive result)
All the best
Hi Theo,
With 300,000 BV2 per well/6 well plate and 14 hours exposure to 1ug/ml LPS I dilute my supernatant 1/10 and run either on IL1B Elisa, or multiplex Elisa. You will see 100's of pg/ml of culture solution. The Elisas available for IL1B are extremely robust. I would look first to how many cells that you have, your LPS (has it worked before), the storage of the samples and wether you feel comfortable with how the kit was run. A western is a good idea or a PCR on the cells you harvested. One will see a massive increase in the gene to pair with the release. Hope that helps. B.
Bobbi,
you think its just a matter of concentration? I want to be careful cause I dont have much S/N left. I ran a qPCR and I know when the gene is overexpressed, but I also want the protein data to pick the point when the protein levels are also high enough. So, 10x u think is enough?
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