Transfecting Plasmid DNA Into NCI-H1299 Cells Using Lipofectamine 3000 Reagent

Proper culture techniques and procedures are an essential part of ensuring successful transfection. Subculturing, also referred to as passaging, is the removal of medium and transfer of cells from a culture into fresh growth medium, in order to propagate the cells.

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Brightfield image of NCI-H1299 cells in culture, prior to transfection.

Complete growth medium

Component	Cat. No.
Gibco RPMI 1640 with GlutaMAX Supplement	61870036
10% Gibco FBS	A3160401
25 mM Gibco HEPES (1 M)	15630080
1.0 mM Gibco Sodium Pyruvate	11360070

Passaging

Maintain cells in T-75 flasks.
Use Gibco TrypLE dissociation reagent.
Passage cells every 3–4 days to ensure that they do not enter senescence.
Transfection of cells should be performed only between passages 5 and 25 post-thaw.
If designing an experiment that involves transfection, ensure that setup coincides with a cell passage.
Plate cells for transfection only 1 day before the experiment.

Seeding cells for transfection

The day before transfection, dissociate cells that are 80–90% confluent in a T-75 flask.
Count the cells using standard trypan blue exclusion.
- Important: The cell number and concentration determined can vary significantly depending on what method is used for counting; it is important to be consistent and use a single method throughout an experiment.
The cell culture must have >90% viability and be 80% confluent on the day of transfection.
- Important: If cells are not at the right confluence, do not wait until the next day to perform transfection, as this can significantly affect transfection efficiency.
Seed 8.3 x 10⁴ cells in 500 μL growth medium for a single well of a 24-well plate.

Transfection protocol

Component	Cat. No.
Invitrogen Lipofectamine 3000 Transfection Reagent	L3000008
Gibco Opti-MEM I Reduced Serum Medium	31985062
Thermo Scientific Nunc 24-Well Cell Culture–Treated Multidish	142475

On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24-well plate using Lipofectamine 3000 Transfection Reagent:

Step	Tube	Complexation Component	Amount per well (24-well)
1	Tube 1	Opti-MEM I medium	25 μL
1	Tube 1	Lipofectamine 3000 reagent	1.5 μL
2	Tube 2	Opti-MEM I medium	25 μL
		DNA amount (DNA concentration should be 0.5–5 μg/μL)	250 ng
		P3000™ reagent	0.5 μL
3	Add tube 2 solution to tube 1 and mix well
4	Incubate mixture from step 3 at room temperature for 10–15 min
5	Add 50 μL of complex from step 4 to cells; gently swirl plate to ensure homogeneous distribution of complex to the entire well

Transfection efficiency analysis

At 48 hr following transfection of a GFP reporter construct, cells were evaluated via microscopy and flow cytometry. To assess transfection efficiency, cells were first visualized via fluorescence microscopy for qualitative assessment of protein expression, morphology, and viability (Figure 1). Cells were then prepared for flow cytometry by aspirating the medium and replacing it with 250 μL of a 7:3 mixture of TrypLE reagent:1X DPBS. Cells were incubated at 37°C for 15 min and then pipetted up and down to ensure single cells for flow cytometry analysis.

Fluorescence images demonstrating 62% transfection efficiency

Figure 1. Post-transfection analysis of cells. (A) Fluorescence and (B) bright-field images demonstrating 62% transfection efficiency.

Tips and tricks

Decreasing the serum content of the culture medium (to <10%) at the time of transfection is acceptable, but replace with complete growth medium within 4–24 hr post-transfection.
Antibiotics can be used during transfection.
Prior to flow cytometry, visualize cells under a bright-field microscope to verify dissociation following incubation with TrypLE reagent.
If cells have not fully detached after 15 minutes, place them back in the incubator for another 10 to 15 minutes.
Gently tap on the side of the plate to aid in cell detachment when observing only partial attachment or detachment time is taking more than 30 minutes.

Scaling up or down Lipofectamine 3000 reagent transfections

Use the following table to scale the volumes for your transfection experiment. The most common sizes are listed below.

Culture vessel	Multiplication factor*	Shared reagents		DNA transfection			siRNA transfection
Culture vessel	Multiplication factor*	Growth medium	Opti-MEM medium for complexing	DNA	P3000 reagent	Lipofectamine 3000 reagent**	siRNA	Lipofectamine 3000 reagent**
96-well	0.2	100 μL	2 x 5 μL	50 ng	0.1 μL	0.3 μL	3 pmol	0.3 μL
48-well	0.5	250 μL	2 x 12.5 μL	0.25 μg	0.5 μL	0.75 μL	7.5 pmol	0.75 μL
24-well	1	500 μL	2 x 25 μL	0.5 μg	1 μL	1.5 μL	15 pmol	1.5 μL
12-well	2	1 mL	2 x 50 μL	1 μg	2 μL	3.00 μL	30 pmol	3 μL
6-well	5	2 mL	2 x 125 μL	2.5 μg	5 μL	7.50 μL	75 pmol	7.5 μL
60 mm	11.05	5 mL	2 x 250 μL	5.5–11 μg	11–22 μL	16.5 μL	166 pmol	17 μL
10 cm	28.95	10 mL	2 x 500 μL	14–28 μg	28–56 μL	43 μL	434 pmol	43 μL
T-75	39.47	15 mL	2 x 750 μL	20–40 μg	40–80 μL	59 μL	592 pmol	59 μL
T-175	92.11	35 mL	2 x 1.75 μL	46–96 μg	92–180 μL	138 μL	1,382 pmol	138 μL
* After determining the optimum reagent amount, use the multiplication factor to determine the reagent amount needed for your new plate format. ** Optimum amount needed is determined from the protocol for Lipofectamine 3000 Transfection Reagent.

Ordering information

Gibco Lung Cancer Starter Kit

For your convenience, the essential components of this protocol are now available in the Gibco Lung Cancer Starter Kit. The kit includes: basal medium, FBS, Lipofectamine 3000 reagent, Opti-MEM medium, and TrypLE reagent. The kit is available at thermofisher.com/cancercellculture. For additional components required for the protocol see ordering table below.

Find out more at thermofisher.com/3000

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