I want to measure cytokine production of infected macrophages using the human CBA inflammation kit (BD), but the cytometer that I want to use is in a P1 room, and I'm not allowed to acquire the samples on it as the pathogens are supposed to also be in the supernatants (P2 needed). I don't know a method for decontaminating the samples that doesn't affect the cytometric assay. I thought about using antibiotics in the samples, but I cannot be sure it's not affecting the assay and I'm very afraid of having the samples one hour at RT. Fixing the supernatants with PFA or something similar seems weird.
Have you been in the same situation? Can anybody help me with some ideas?
Did you think about filtrating your samples? Alternatively, i would probably use gentamycin to eliminate any extracellular bacteria after your infection period.
Have you considered using another detection method? If you are attempting to screen for qualitative changes in various cytokines, you could use R&D systems cytokine array kits. They are essentially spot blot westerns with pre-labeled antibodies on a nitrocellulose membrane. Or if you have one or two specific cytokines you are interested in, you could just perform a basic quantitative ELISA. Both of these you could do in your P2 lab, and the contamination should not affect either.
You could always run your samples through a low-binding syringe filter as well, as this is used for sterilization of tissue culture media and such, but you may lose a lot of your sample in the process and it may not be reproducible between samples.
Overall, unless you can find a flow cytometer that allows infected cells, you may need a new detection method. Good luck.
Hi I beleive you are using the following kit (http://www.bdj.co.jp/pdf/64-01-81014-551811-C_web.pdf).
However a wide variety of microbes will die with sodium azide. I believe your wash reagents in that kit will have sodium azide (check the kit manual). If not you can fix cells with fixatives like formaldehyde and analyse the samples.
I shall get you NUS, Singapore, Flowcytometry unit head E.mail ID to assist you further in a due course.
Did you think about filtrating your samples? Alternatively, i would probably use gentamycin to eliminate any extracellular bacteria after your infection period.
Did you consider the use of UV ? We usually use 30 min of UV to inactivate supernatant containing viruses.
I have also used the CBA for detection of cytokines and chemokines from supernatants of cell samples or homogenised tissue infected with various viruses and fungi (Not Human though). I also contacted BD with this problem about having to use Cat1 (PC1) FACS facilities. The answer was to perform the CBA as normal in your PC2 lab and then fix the samples at the end of the assay prior to running them on the FACS (I used PFA). This seemed to work and did not diminish the signal from the assay compared to unfixed standards. Importantly you fix after the assay and not the samples prior. It is always good to re-test samples with an ELISA as we have found that some of the CBA's under estimate the quantity of the cytokine but not the general trend when comparing across samples.
I agri with Mr.Paul Whitney. you can use paraformaldehyde to fix the cells. Normally researchers use 4% paraformaldehyde to fix the cells. I hope this is sufficient to avoid bacterial contamination in the supernatnant.
Dr.Paul Edward Hutchinson ([email protected]),
you can contact him reg your problem.
I would largely concur with Margherita. I regularly conduct ELISAs on supernatants after challenging with pathogenic bacteria. In my assays I use gentamicin for 21 hours to kill the bacteria, which I know works from viable counts. Everything is compared to the untreated samples to control for the effect of the antibiotic on the host cells, however it is somewhat controversial in some quarters as this is not truly representative on the environment. Otherwise I would filter sterilise the supernatants with a 0.2um filter in a Class II hood. Or centrifuge, then filter if there is a high concentration of bacteria in the supernatants. I know of some groups in Imperial College who use this method.
Good luck Pedro.
I had to analyze supernatants from M. tb infected macrophages so BSL3 samples, not using CBA but 30-plex luminex. I used 0.2um 96 well filter plates. Worked really well for me.
Many thanks to all of you!
BD recommends me:
"You can fix with PFA 1% during 20 min à 4°C, then you wash and resuspend beads in usual buffer ". Same than Paul Whitney said. I will try this.
UV treatment over ice and the existence of Sodium Azide in my washing buffers are also good options to convince the cytometer manager to allow me to pass the samples. Gentamycin treatment of the supernatants seems no good: very long time of the supernatants at 37°C. Filtrating is not a good option as I have only 50ul of sample and for sure will loss some volume.
Thank you very much and best regards!!!
What I would no do at all is passing the samples through a 0.22 filter. I've done it before and the samples were negative of IL-8 by ELISA while if I ran the samples without filtering, they were super positive. I can't tell it happens, but it seems the filter binds a lot of proteins
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