I am hoping to find some direction or options for the following issue:
Issue started with only one research (out of 7) and spread to all researchers over time. When transfecting (have tried Lipo3000, Lipofectamine, Lipo2000, and Fugene 4K) any cell line (tried new and current of A549, Vero, Hep2, and 293Ts) on coverslips (tried 6 brands and types) for IF with the standard protocol, as well as several experiments changing proportions (guided directly by Thermo technical support), we consistently see small dots all over the slide. When we do a mock transfection with Optimem instead of DNA, there is no issue. Viral infections also do not have the issue. Additionally, we have tried new FBS batches, new Optimem, fresh reagents, fresh media, professionally prepped DNA, multiple vector types, myco tests (cell lines all negative), extra washes with PBS before and after fixation, filtering DNA with a .22um syringe filter, fresh mini/maxi prep kits (MN), other brands of plasmid prep kits (nanodrop readings clean, but lower than usual yields), benzonase treatment post fixation, as well as other tests. The issue has gone on for a while and we are grasping at straws.
It is also important to note that methods used for this protocol were established many years before issue with no problems. Also, that other experiments using transfections are not working, but they don't have a visual indicator like DAPI in IF. I believe the Lipo complexes are unable to enter the host cells. When using GFP plasmids some cell signal is seen after 24H, but is far lower than usual.
Any thoughts or ideas are welcome.
https://www.researchgate.net/post/Small-DAPI-dots-blue-specks-in-transfected-cells-What-can-I-do-to-avoid-this/2
Here is one additional photo of a transfected sample that had the contamination very bad, may be helpful.
Hi Kearstin Edmonds
Can I ask you the batches of media/ reagents that you are using are new productions ? After summer 2022 ?
Noemi Asfogo, we have tested 2 brands of FBS, total 3 batches. Also, 2 brands of DMEM, 3 formulas and several batches. Issue began in Spring 2022. We have tracked down that there is something environmentally causing general cell health issues, which results in increased transfection toxicity, and as such transfection reagents that do not enter cells and stick to coverslips, causing this DAPI staining. We used all of our reagents in a different lab/hood/incubator and the issue was not present. We took cells from that same lab into our environment and they began to have the issue, and it worsened over a couple of weeks. I replaced incubator HEPAs, gas inlet filters, ran a steri-cycle, fresh sterile distilled water, added Aquaguard for incubators, and calibrated CO2 levels. I am transfecting today to test if those changes had any impact to cells over the past week.
Thank you for your response and any thoughts are appreciated!
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