Transfecting Plasmid DNA Into MCF10A Cells Using Lipofectamine 3000 Reagent

Complete growth medium

Component	Cat. No.
Gibco Ham’s F-12 Nutrient Mix	11765054
5% Horse Serum	–
1 mM Gibco Sodium Pyruvate	11360070
25 mM Gibco HEPES	15630106

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. See our product list for this protocol

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 2 x 10⁵ cells in 500 μL growth medium for a single well of a 24-well plate.

Transfection protocol

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 Invitrogen Lipofectamine 3000 Transfection Reagent:

Step	Tube	Complexation components	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)	0.5 μL
		P3000™ reagent	1 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 10 min and then pipetted up and down to ensure single cells for flow cytometry analysis.

Figure 1. Post-transfection analysis of cells. (A) Fluorescence and (B) bright-field images demonstrating 10–20% 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.

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	0.1 µg	0.2 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 µL	30 pmol	3 µL
6-well	5	2 mL	2 x 125 μL	2.5 µg	5 µL	7.5 µL	75 pmol	7.5 µL
60 mm	11.05	5 mL	2 x 250 μL	5.5–11 µg	11–22 µL	17 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 mL	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.

Find out more at thermofisher.com/3000

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For Research Use Only. Not for use in diagnostic procedures.