As title, my sample is kind of plant-based protein(pI=4.5) and its hydrolysates and glycated protein.
I treat my sample with CACO-2 cells in MTT test, but I found that my sample is ph-sensitive, it always precipitates even I centrifuge it and pass through 0.22um filter before adding to cell.
I guess when I dilute the sample solution (protein well dissolved in pH 7.0 PBS buffer) with free DMEM (medium in room temperature and normal environment it pH near to 8.0 and show as pink-purple), it is all good, but after I adding to cells and send my plates back to CO2-controlled incubator, its pH value get lower, maybe 7.2 to 7.4 and shows red-orange color, my protein sample in DMEM is starting to partial precipitate..., and the next day washing each cell wells I can see the turbidy residue on the cell culture. Also, it effects my MTT results (Higher concentration will have too high viability like 120-150%, and lower concentration will get low viability, and I thought it is because of my unstable samples hamper cell growth).
How can I avoid this bad situation when my sample is easy to precipitation when pH changes?
Should I directly dissolved my sample in DMEM? But after the medium back to the incubator it will also lower the pH again.
Thank you for your patience in reading my question. Really need some advices and help...
Dear Zhao Liang Weng
you can try to use the XTT assay instead the MTT.
XTT is a soluble derivate of MTT which is secreted outside the cells in viable cells and the intensity of colourn on the surnatant could be correlated with the number of live cells.
https://www.sigmaaldrich.com/IT/it/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-counting-and-health-analysis/cell-proliferation-kit-xtt-assay
one other nice alternative could be the Cell titer blue which is a resofulin based assay, which is more sensitive than XTT since the metabolite is fluorescent and signal could be detected using either a plate-reading fluorometer (preferred) or spectrophotometer.
https://ita.promega.com/products/cell-health-assays/cell-viability-and-cytotoxicity-assays/celltiter_blue-cell-viability-assay/?catNum=G8080
good luck
Manuele
Did you observe the same effect in non-cultured conditions?
I mean do you able to dissolve the protein-based sample in pH approx 7.2-7.4 buffer?
Can you also confirm that precipitate and pH change is not caused due to cell culture contaminants (yeast, bacteria, mold, virus etc..)?
pI 4.5 must be still solubilized in pH 7 roughly if other considerations, such as heat-induced aggregation, conformational changes, oligomerization, denaturation/unfolding, or chemical modification, etc...
Is your sample pure protein or some vegetal components have also been accompanying?
Test the gradually decreased pHs alone with your POI to see only the pH effect first, then we may diagnose the problem and propose solutions...
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