Hi Everyone! We are trying to test mast cell degranulation in RBL2H3 cell line by measuring beta hexoaminidase in the supernatant. We are unable to detect the enzyme in the supernatant nor in the cell lysate. Cells are sensitized with IgE (mice anti-DNP) and with antigen DNP-BSA promoting degranulation. We didn't get any detectable enzyme in the sup so we tested cell lysate but got nothing (from the staining, granules are present). Antibody and DNP-BSA are from Sigma. Substrate
(p-Nitrophenyl-N-acetyl-β-D-glucosaminide) for the enzyme is from Millipore. We stained the cells and observed the granules after the IgE and Antigen treatments and we are not seeing degranulation. Anyone with advice? Thank you!
Hi, it could be several things-buffer pHs (most likely), unhealthy cells, IgE or antigen not good / wrong concentration, lysis detergent wrong concentration. We use Tyrode's buffer pH 7.4, substrate=4.5, and stop solution=10.7. Even if you made the buffers at the correct concentration, the Tyrode's buffer pH could change over time, even with a buffering agent, so I recommend checking the pH before every experiment. Generally 1ug/mL IgE and 100ng/mL DNP-BSA are good, although the valency (number of DNPs per BSA molecule) could change these figures. 1% v/v Triton-X is good for lysis. Generally 1 hour stimulation is good and consistent. You may see some morphological changes, but don't expect to see complete degranulation using IgE and antigen. If you're using SPE-7 IgE, be careful as it can activate cells without antigen.
Thanks, Daniel. Could I ask you couple of questions about the buffers? I believe we used properly pH'd buffers but we used DMEM (with 15% FBS) for stimulations and citrate buffer pH 4.5 with substrate. We used DMEM with FBS because we maintain the cell line in it. Do you use any FBS in your Tyrode's buffer? What dilution buffer do you use for your Triton X? I think we used PBS or water. Thanks again for your help! It really helps! :)
I wouldn't use DMEM+FBS for stimulations. The main problem is that we usually measure absorbance at 405nm for the beta-hex assay, and FBS also absorbs at this wavelength so your background would be really high. Also, the pH of DMEM will increase beyond 8 or 9 outside of the CO2 incubator while you're doing pipetting (if there's phenol red you'll see it turn pink). Most people use HEPES-buffered Tyrode's buffer and that's what I recommend (don't add FBS for the above reason, but you can add 1% BSA). I think Triton X in PBS is ok as long as the pH is the same as the stimulation buffer, but I'd prefer to make it in Tyrode's buffer for consistency reasons.
If you insist on using DMEM, maybe you can try DMEM without FBS, and adding 500mM HEPES pH 7.4 to 25mM final. I don't think anything in DMEM (even phenol red) absorbs at 405nm so it may be possible, but I've never tried this...
I sure will try Tyrode's buffer and what you suggested above. If you don't mind me asking one last (hopefully) question, we are planning on trying A23187 as a positive control. I was wondering if you use any other degranulating stimulator (eg. calcium Ionophores) as a control. If so, do you add exogenous calcium to your media (at a dose of)? Thank you very much for your advice and time! Thank you!
A23187 works well at 1ug/mL. Tyrode's buffer (and DMEM) have 1.8mM Ca2+, which should be sufficient. My chemistry is awful and I don't know what happens if you add more calcium chloride, but from my transfection experience I think it can react with sodium phosphate to make calcium phosphate precipitates, although media formulations tend to have both of the former?? Maybe someone else can clarify? But even if you managed to add more calcium without causing precipitation, I think background activation will increase. Personally I wouldn't add any more calcium...
Dear Hyo, has your RBL-2H3 problem been solved? I cannot induce degranulation (beta-hex) using anti-DNP IgE and DNP-BSA either, but I am sure granules are inside and I could detect a high level of beta-hex by cell lysis or ionomycin stimulation. Very looking forward to your reply. Thanks.
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