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

Brightfield image of Calu-6 cells in culture prior to transfection

Brightfield image of Calu-6 cells in culture, prior to transfection.

Complete growth medium

Component Cat. No.
Gibco RPMI 1640 with GlutaMAX Supplement 61870036
10% Gibco FBS A3160401
0.1 mM Gibco MEM NEAA Solution 11140050
1.0 mM Gibco Sodium Pyruvate 11360070


  • 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 1.1 x 105 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
Lipofectamine 3000 reagent 2 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 10 min and then pipetted up and down to ensure single cells for flow cytometry analysis.

 Posttransfection analysis of Calu-6 cells fluorescence images
 Posttransfection analysis of Calu-6 cells bright-field images

Figure 1. Posttransfection analysis of cells. (A) Fluorescence and (B) bright-field images demonstrating 48% 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 posttransfection.
  • Antibiotics can be used during transfection.
  • Prior to flow cytometry, visualize cells under a brightfield 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 Multipli-cation factor* Shared reagents DNA transfection siRNA transfection
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 0.4 μL 3 pmol 0.3 μL
48-well 0.5 250 μL 2 x 12.5 μL 0.25  μg 0.5 μL 1 μL 7.5 pmol 0.75 μL
24-well 1 500 μL 2 x 25 μL 0.5 μg 1 μL 2 μL 15 pmol 1.5 μL
12-well 2 1 mL 2 x 50 μL 1 μg 2 μL 4 μL 30 pmol 3 μL
6-well 5 2 mL 2 x 125 μL 2.5 μg 5 μL 10 μL 75 pmol 7.5 μL
60 mm 11.05 5 mL 2 x 250 μL 5.5–11 μg 11–22 μL 22 μL 166 pmol 17 μL
10 cm 28.95 10 mL 2 x 500 μL 14–28 μg 28–56 μL 58 μL 434 pmol 43 μL
T-75 39.47 15 mL 2 x 750 μL 20–40 μg 40–80 μL 79 μL 592 pmol 59 μL
T-175 92.11 35 mL 2 x 1.75 mL 46–96 μg 92–180 μL 184 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 For additional components required for the protocol see ordering table below.