- It is recommended to allow microemulsion bubbles to settle on
the imaging surface (cover glass) for a few minutes before
imaging. - Microemulsions should be prepared fresh and used within
hours after preparation. - We have found that after microporation HeLa cells adhere
faster and spread better on fibronectin-coated surfaces. - To achieve high transfection efficiency and cell viability, it is
critical that cells are well-spread and not overconfluent before
being subjected to microporation experiments. - The flask should be incubated at 37C for enough time to
ensure all of the cells are fairly rounded and detached from the
surface. - To achieve high cell viability after microporation, gentle pipet-
ting to minimize shearing of the cells is critical. - The number of cells used per microporation is cell line-specific
and may affect the transfection efficiency and viability. A
detailed list of cell line and the number of cells to use per
microporation can be found on the Neon transfection system
website: https://www.thermofisher.com/us/en/home/life-
science/cell-culture/transfection/transfection-selection-
misc/neon-transfection-system.html. - It is important to wash the cells with enough media to remove
unincorporated probes, which can contribute to background
fluorescence and hamper accurate fluorescence quantification. - After every wash, it is recommended not to aspirate out the
entire wash media as this risks aspirating out the loose cell pellet
formed by the small number of cells. - Prior to seeding the cells into the 8-well chamber, it is critical
to wash the chamber with 1PBS to remove unbound fibro-
nectin. Residual unbound fibronectin can inhibit cell attach-
ment to the surface. - We have found that taking multiple ROIs around the object
gives more accurate assessment of the total background fluo-
rescence within the object ROI. - To determine the extent of MB nonspecific opening in cells
based on fluorescence measurements acquired in solution and
in cells, it is important to make sure that the emission proper-
ties of the MB reporter dye and reference dye are the same in
solution and in living cells. We have previous published a
method for assessing the sensitivity of commercially available
fluorophores to changes in environment [20]. - We have shown that the 2Me/PSLOOPMB was the most stable
configuration in other cell types including HEK-293, Jurkat,
Optimizing Molecular Beacons 255