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-H520 cells in culture, prior to transfection

Brightfield image of NCI-H520 cells in culture, prior to transfection.

Complete growth medium

ComponentCat. No.
Gibco RPMI 1640 with GlutaMAX Supplement61870036
10% Gibco FBS A3160401
25 mM Gibco HEPES15630080
1.0 mM Gibco Sodium Pyruvate11360070


  • 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 60% 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

ComponentCat. No.
Invitrogen Lipofectamine 3000 Transfection ReagentL3000008
Gibco Opti-MEM I Reduced Serum Medium31985062
Thermo Scientific Nunc 24-Well Cell Culture–Treated Multidish142475

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:

StepTubeComplexation ComponentAmount per well (24-well)
1Tube 1Opti-MEM I medium25 μL
Lipofectamine 3000 reagent1 μL
2Tube 2Opti-MEM I medium25 μL
DNA amount (DNA concentration should be 0.5–5 μg/μL)500 ng
P3000™ reagent1 μL
3Add tube 2 solution to tube 1 and mix well
4Incubate mixture from step 3 at room temperature for 10–15 min
5Add 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 NCI-H520 cell fluorescence images
Posttransfection analysis of cells NCI-H520 bright-field image

Figure 1. Posttransfection analysis of cells.(A) Fluorescence and (B) bright-field images demonstrating 55% 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 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 vesselMultipli-cation factor*Shared reagentsDNA transfectionsiRNA transfection
Growth mediumOpti-MEM medium for complexingDNAP3000 reagentLipofectamine 3000 reagent**siRNALipofectamine 3000 reagent**
96-well0.2100 μL2 x 5 μL

100 ng

0.2 μL0.2 μL3 pmol0.3 μL
48-well0.5250 μL2 x 12.5 μL0.25  μg0.5 μL0.5 μL7.5 pmol0.75 μL
24-well1500 μL2 x 25 μL0.5 μg1 μL1 μL15 pmol1.5 μL
12-well21 mL2 x 50 μL1 μg2 μL2 μL30 pmol3 μL
6-well52 mL2 x 125 μL2.5 μg5 μL5 μL75 pmol7.5 μL
60 mm11.055 mL2 x 250 μL5.5–11 μg11–22 μL11 μL166 pmol17 μL
10 cm28.9510 mL2 x 500 μL14–28 μg28–56 μL29 μL434 pmol43 μL
T-7539.4715 mL2 x 750 μL20–40 μg40–80 μL40 μL592 pmol59 μL
T-17592.1135 mL2 x 1.75 mL46–96 μg92–180 μL92 μL1,382 pmol138 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.