Hi guys,
I isolate P0-P3 primary microglia by magnetic column method and we get about 1 million cells from each pup.
I used Thermo fischer Lipofectamine 3000 for transfection. Here are the numbers for the same:
for a 12-well plate -
microglia - 0.4 million/ well
Lipofectamine - 3ul / well
p3000 - 2.5 ul /well
DNA - 1.25 ug / well
Incubation before adding - 15mins
Plated cells on Day 1 with PLL and transfection on Day 2. I did florescence imaging on Day 4 (48hrs later)
And we got
Some cell types are notorious for being difficult-transfect. One of the things i would recommend is mycoplasma testing as it can severely impact transfection effeciency without showing the signs of contamination (pH change, cloudiness, etc). In my doctorate thesis, i encountered a similar problem with Lipofectamine 2000 for cultured cardiomyocytes which are also difficult to transfect. There are some parameters that you can "optimize" to boost your transfection effiency. See 5.2.3 Transfection of my thesis:
https://epublications.bond.edu.au/cgi/viewcontent.cgi?article=1195&context=theses
Another alternative would be to generate "stable" cell lines using a cytotoxic reagent but this is no easy task either. Viral options could be another approach.