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I need to optimize a transfection protocol to transiently express a plasmid encoding a chimera of eyfp attached to the c term of a Golgi apparatus signaling molecule) in hela cells and hepg2 cells and get as high expression as I can get.

I need enough protein for about 100 wb's. I've seeded hela cells to 12 150mm dishes. And the same with the hepg2. I've prepped mg's of plasmid.

I'm using invitrogens original lipofectamine reagent; not the 2000, or ltx or any of that stuff.

Any suggestions on a protocol here for hela and hepg2 plasmid dna and lipofectamine mediated transfection on such a large plate size.

I figure just scaling up should work but there's some pitfalls here. Look at this table for example when you go from 60 to 100 mm you about double the area, so you also about double the culture volume. However when you go from 100 to 150mm you triple the area, but only increase the culture volume two fold about. This is making my scale-up calculations for transfection look really off:

culture volumes and areas

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    $\begingroup$ Despite what the numbers look like, across all values going from 100mm to 150mm you're scaling up by a factor of ~2.76: (a) 152/55 = 2.76, (b) 1.52e7/5.5e6 = 2.76, (c) |avg(30.4-45.6)/avg(11-16.5)| = 2.76. $\endgroup$
    – CKM
    Mar 26, 2015 at 0:18
  • $\begingroup$ Ahhh! I should have crunched the numbers cause you R correct. It's a weird concept because when you scale up in vessel size you want all parameters, ideally I would think, to increase at the same rate. However the depth of media can reach a certain point where I believe it hinders growth. When the volume gets to be too much it can take too long for those needed soluble factors to build up in the environment. Also increase in medium depth can delay delivery of soluble oxygen to the cells. Thank you for seeing through $\endgroup$
    – rhill45
    Mar 26, 2015 at 0:34
  • $\begingroup$ All my nonsense to make that comment. I think this ? Is a little scatter I'm going to reword it to focus it on the ratio, area and volume if you want to post an answer its yours. I'll post a seperate ? On the hela optimization $\endgroup$
    – rhill45
    Mar 26, 2015 at 0:35
  • $\begingroup$ @Kendall please post your comment as an answer to close this question. Thanks! $\endgroup$
    – cagliari2005
    Mar 29, 2015 at 6:05

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So, for every row on the dish protocol, the entire process is scaled up a discrete value. In the case of 100mm to 150mm, the values are scaled up by about 2.76 across all values:

(a) 152/55 = 2.76, (b) 1.52e7/5.5e6 = 2.76, (c) |avg(30.4-45.6)/avg(11-16.5)| = 2.76

(as answered in comments)

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