Been doing beta-hexosaminidase release assay but the results hasn't been promising. Firstly, there's no colour development in the supernatant and secondly the release has been below 10% for all groups.
Tried doing ELISA but there wasn't any difference between the normal and induced groups.
What went wrong - the cells, the antibody, buffer??
Dear Audrey,I have never done this assay, then I do not add any specific answer. However,some general considerations can be done:
1) Are you sure that the volume where you measure the enzymatic activity is as low as the assay of small amount of enzymatic activity requires to be tested?
2) You do not report any data on the type of cells you are investigating. Are you sure that they are the best cells to use for the assay?
3) Are you sure there is no inhibitor of the enzymatic activity in the supernatant?
I hope these questions can help you, Angela
Hi Angela, thank you for your responses.
1. This assay was done by my senior previously and his results were around 30%. From a journal I read recently, it was reported that the percentage release is around 50-60%.
2. I am using RBL cells which is the ideal cell line for this study.
3. What inhibitor are you referring to? I doubt there is any.
I've done beta-hex assays with RBLs for years. There are many potential problems, but pH is the most likely. RBLs are unresponsive at low pH. If you're using Tyrode's buffer the pH should be close to 7.4. Even with HEPES I've noticed that the buffer pH will decrease over time if the buffer isn't frozen. I used to check the Tyrode's buffer pH every time I did the assay. I've never seen the substrate (pH 4.5) or stop solution (pH 10.7) go off pH. This could explain your ELISA result as well if you used the same buffer. What cytokine did you assay for?
Hi Daniel, thank you for your input. Managed to sort out the beta-hexo assay, translated the same conditions to ELISA. It worked when we're optimising the TNF-alpha release time but when we treated the cells with our treatment, the cells did not respond again.
- Will the pH of Tyrode's buffer change within a month?
- Does incubating the cells in Tyrode's buffer for a long period of time ie. 10 hours affect the cytokine release?
- Does the amount of glucose in the Tyrode's buffer play a factor if the cells were to be incubated for prolonged time? The current recipe uses 1g/L of glucose.
Hi Audrey, I didn't realise you were doing ELISA and beta-hex on the same supernatant! I haven't tried Tyrode's buffer for 10 hours, but I have a feeling it's not optimal for the cells, even with extra glucose. Can you do separate assays? Beta-hex with Tyrode's and TNF alpha with culture medium? To your first question: only way to know is the check the pH often. I usually freeze 50mL aliquots of Tyrode's buffer so newly defrosted aliquots should be fine. 4C for a month may or may not be ok.
Hi Daniel, sorry for the confusion, the assays were done separately.Previously, a senior has done ELISA using Tyrode's buffer for 20 mins treatment followed by 6 hours induction which worked fine. However, I shall take your suggestion of using culture medium instead.
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