- The number of cells used per microporation is cell type-specific
and may affect transfection efficiency and viability. A detailed
list of cell types and the number of cells to use per micropora-
tion 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. - Avoid introducing air bubbles during pipetting, as they can
cause sparks during microporation that reduce transfection
efficiency and cell viability. - We tested a range of RBMB concentrations (0.05–4.8μM). We
found 0.8μM to be an optimal concentration. - Microporation parameters are cell type-specific. See the Neon
Transfection system website (seeNote 6) for optimal para-
meters for each cell line. - As untransfected probes can emit background fluorescence and
thus hamper sensitive RNA detection, make sure that after
microporation cells are sufficiently washed with enough
media to remove untransfected probes. - After each wash, it is recommended not to aspirate out all of
the wash media as this may increase the risk of aspirating out
the loose cell pellet formed by the small number of cells. - Analysis can be performed using Fiji or ImageJ. Fiji can be
downloaded at:http://fiji.sc/. ImageJ can be downloaded at:
https://imagej.nih.gov/ij/. - In our experience, setting rolling ball radius to 2.0 pixels can
enhance particulate objects without losing too much informa-
tion for RNA analysis. It is recommended that the operator
tries different parameters to find an optimal value. - The background can be further subtracted from the ROI of the
cell. This could be achieved by measuring the pixel intensity of
several ROIs outside the cell and subtracting the average value
from the total image using Process>Math>Subtract. - The LoG3D plugin can be downloaded from:http://bigwww.
epfl.ch/sage/soft/LoG3D/. To activate, place the file in the
‘Plugin’ folder of Fiji or ImageJ. - Images appear in gray scale after processing.
- In our experience, these parameters enable accurate identifica-
tion of particulate objects. It is recommended that the operator
tries different parameters to find optimal values for their
microscopy setup. - The ‘FindStackMaxima.ijm’ file should be placed in the Macros
folder of Fiji or ImageJ. Install the file by selecting Plugins>
240 Yantao Yang et al.