Transfection Optimization

Because transfecting a large number of plasmids (~8) into HEK293 cells can drastically increase cytotoxicity and lower transfection efficiency, we are optimizing our transfections before we start characterizing the B-Cell Receptor. We plan on evaluating suspended vs. adherent transfection and varying total mass of DNA transfected.

Approach

We will be testing in duplicate 10, 25, 50,100, 250, 500, and 1000ng of DNA with lipo 3K suspended vs non suspended transfection to determine optimal transfection conditions for our cells.

Parts Needed

Part	Status
hEF1a:rtTa
TRE:Gal4VP16
UAS:mKate
hEF1a:eYFP
hEF1a:eBFP

Results:

Gating

Adherent Transfection

	Untransfected	10ng	25ng	50ng	100ng	250ng	500ng	1000ng
	HEK293	hEF1a:rtTA *2ng* TRE:Gal4VP16 2ng UAS:eYFP *2ng* hEF1a:mKate *2ng* hEF1a:eBFP *2ng* 1000 nm Dox	hEF1a:rtTA 5ng TRE:Gal4VP16 5ng UAS:eYFP 5ng hEF1a:mKate 5ng hEF1a:eBFP 5ng 1000 nm Dox	hEF1a:rtTA 10ng TRE:Gal4VP16 10ng UAS:eYFP 10ng hEF1a:mKate 10ng hEF1a:eBFP 10ng 1000 nm Dox	hEF1a:rtTA 20ng TRE:Gal4VP16 20ng UAS:eYFP 20ng hEF1a:mKate20ng hEF1a:eBFP 20ng 1000 nm Dox	hEF1a:rtTA 50ng TRE:Gal4VP16 50ng UAS:eYFP 50ng hEF1a:mKate 50ng hEF1a:eBFP 50ng 1000 nm Dox	hEF1a:rtTA 100ng TRE:Gal4VP16 100ng UAS:eYFP 100ng hEF1a:mKate 100ng hEF1a:eBFP 100ng 1000 nm Dox	hEF1a:rtTA 200ng TRE:Gal4VP16 200ng UAS:eYFP 200ng hEF1a:mKate 200ng hEF1a:eBFP 200ng 1000 nm Dox
Red vs. Blue
Yellow vs. Blue
Co-Transfection Efficiency	0%	0%	0.053%	0.12%	0.74%	4.33%	5.37%	3.15%
Red vs. Blue
Yellow vs. Blue
Co-Transfection Efficiency	0%	0%	0.047%	0.29%	1.23%	5.87%	5.62%	2.91%
Co-Transfection Efficiency Average	0%	0%	0.05%	0.205%	0.985%	5.1%	5.945%	3.03%

Suspended Transfection

	Untransfected	10ng	25ng	50ng	100ng	250ng	500ng	1000ng
	HEK293	hEF1a:rtTA *2ng* TRE:Gal4VP16 2ng UAS:eYFP *2ng* hEF1a:mKate *2ng* hEF1a:eBFP *2ng* 1000 nm Dox	hEF1a:rtTA 5ng TRE:Gal4VP16 5ng UAS:eYFP 5ng hEF1a:mKate 5ng hEF1a:eBFP 5ng 1000 nm Dox	hEF1a:rtTA 10ng TRE:Gal4VP16 10ng UAS:eYFP 10ng hEF1a:mKate 10ng hEF1a:eBFP 10ng 1000 nm Dox	hEF1a:rtTA 20ng TRE:Gal4VP16 20ng UAS:eYFP 20ng hEF1a:mKate20ng hEF1a:eBFP 20ng 1000 nm Dox	hEF1a:rtTA 50ng TRE:Gal4VP16 50ng UAS:eYFP 50ng hEF1a:mKate 50ng hEF1a:eBFP 50ng 1000 nm Dox	hEF1a:rtTA 100ng TRE:Gal4VP16 100ng UAS:eYFP 100ng hEF1a:mKate 100ng hEF1a:eBFP 100ng 1000 nm Dox	hEF1a:rtTA 200ng TRE:Gal4VP16 200ng UAS:eYFP 200ng hEF1a:mKate 200ng hEF1a:eBFP 200ng 1000 nm Dox
Co-Transfection Efficiency 1	0%	0%	0.063%	0.19%	1.07%	4.61%	3.95%	2.3%
Co-Transfection Efficiency 2	0%	0%	0.066%	0.32%	0.94%	4.3%	4.77%	2.23%
Co-Transfection Efficiency Average	0%	0%	0.0645%	0.255%	1.005%	4.455%	4.36%	2.265%

x-axis label	ng
1	0
2	10
3	25
4	50
5	100
6	250
7	500
8	1000

*performed a T-test on data (albeit sketchy T-test, given that there were only 2 duplicates of each condition) --> suspended vs. adherent show no significant difference in co transfection efficiency

Discussion:

Bleed-through:

We should run single color controls to account for bleed-through. The gating that gave the above data excluded a population when gating in the yellow vs blue space on account of bleed through. However, we can't know for sure that that population is just bleed through without single color controls.

Non-linear plot:

y is just y=t
x=0.1t+2

So early on the +2 dominates, but at the higher domain the 0.1t dominates. That is why the plot looks like it does. All a*x vs b*y linear functions are straight diagonal lines. ax+c looks like ax for ax>>c, and like c for ax<<c. and a constant makes a vertical lines on log log axis. That is why it starts pointing up then tilts over and makes a straight diagonal.

Child pages

Transfection Optimization

Results:

Discussion: