They were observed under 40x after treating 3T3L1 pre-adipocyte cells with pterostilbene drug conjugate at 100micromolar concentration. Does apoptosis or necrosis render cells like this or is it something else?
I have observed this form of crystalline structures before, when I was performing drug screens.
then they were formed by the compounds that wouldn't dissolve in aquous conditions in the concentrations I was using them in (50uM), so my best guess is that this is the same problem.
try low concentrations?
at this point you have no clue what the effective concentration in the medium is, as you have no clue what amount of compound is captured in one of those crystals.
I have observed this form of crystalline structures before, when I was performing drug screens.
then they were formed by the compounds that wouldn't dissolve in aquous conditions in the concentrations I was using them in (50uM), so my best guess is that this is the same problem.
try low concentrations?
at this point you have no clue what the effective concentration in the medium is, as you have no clue what amount of compound is captured in one of those crystals.
Thank you so much for replying Arnoud Sir. I will try to work with lower doses now.
Upvoting Arnoud's answer, they indeed look like non-cellular structures (crystals) propably caused by slow crystalization of the undissolved compound. If you have material to spare, you can try to verify this hypothesis by preparing a flask/well with your full medium (drug added of course) minus the cells and see if they form.
Apoptosis or necrosis will give you cellular debris that look much different than what you see in the picture (i could describe them as mostly rounded particles suspended in your flask/dish)
Thanks a lot Theofanis for your suggestion. I can try this tomorrow:D
Surprisingly, the structures in my picture resemble the crystals formed after MTT assay! I am wondering why:/
https://www.google.co.in/search?biw=1366&bih=643&noj=1&tbm=isch&sa=1&q=MTTcrystals&oq=MTTcrystals&gs_l=img.3...8967.11340.0.12952.8.8.0.0.0.0.131.821.4j4.8.0....0...1c.1.64.img..0.2.234.N95tcSwwv9w#imgrc=GP00Dg5IZa5GFM%3A
If you have access to a Dynamic Light Scattering instrument (DLS) perform a concentration series of your compound in PBS or HBSS. You will be able to see at which concentrations the aggregates form. Ideally, using the cell medium as a diligent would be better, but this has FBS or other components which would mask the compound signal due their nature, meaning their particles will also be detected in DLS. If compound should be dissolved in DMSO and then diluted in medium, make sure you have the same final DMSO concentration throughout your PBS or HBSS dilutions. In drug discovery compound aggregates can cause false positives in all assays. Good Luck!
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