The Cignal Transfection Optimization Plate is a tool for quickly optimizing
transfection conditions prior to using Cignal Finder Reporter Arrays. The
Transfection Optimization Plate allows users to determine:
- Optimum transfection reagent
- Optimum cell density
- Amount of transfection reagent required for the assay
Each plate is provided with pre-dispensed controls as described below:
Columns 5 to 12 contain Positive Control reporter, a mixture of a constitutively expressing GFP construct, constitutively expressing firefly luciferase construct, and constitutively expressing Renilla luciferase construct (20:1:1).
Columns 1 to 4 contain empty wells without any pre-dispensed control. Columns 3 and 4 are recommended for mock transfection (or transfection reagent alone) to determine if there is any effect of the transfection reagent on cell viability. Columns 1 and 2 are recommended for untreated cells and serves as 100% viability control.
The protocol follows a simple reverse transfection procedure shown below:
Protocol (example uses adherent cells, but is also compatible
with suspension cells)
- Add 50 µl of cell culture media to each well of the Transfection
Optimization plate. Resuspend the reporter assay constructs by gently
tapping the side of the plate, while slightly rocking the plate back and
forth, then left to right, five times each and incubate it for 5 minutes
at room temperature.
- Add varying amounts of transfection reagent in 50 µl of cell culture
media per well for each individual transfection.
- Follow guidelines of transfection reagent for appropriate incubation
times before addition to each well.
- Add transfection reagent to wells except columns 1 and 2.
Transfection condition should be tested in four technical replicates. Mix
by gently tapping the sides of the plate for at least 30 seconds and
incubate according to transfection reagent protocol guidelines.
- Meanwhile, wash cells in a culture dish once with PBS without calcium
and magnesium, and treat with 1-3 ml trypsin-EDTA for 2-5 minutes at 37ºC
in a humidified atmosphere containing 5% CO2. Suspend the cells in 7-9 ml
of Opti-MEM® containing 5% of fetal bovine serum, then centrifuge the
cells down, remove the supernatant, and resuspend the cells to 6 x 105
cells/ml in Opti-MEM® containing 10% of fetal bovine serum and 1% NEAA.
To ensure reproducible transfection results, it is important to accurately
measure the cell density with a hemacytometer or an automated cytometry
- After the incubation that forms transfection reagent - DNA complexes is
completed, mix the cell suspension by several inversions of the tube
containing the cells or by gentle pipeting of the cell suspension.
- Add pre-determined numbers of cells to each well containing transfection
reagent - DNA complexes. This gives a final volume in each well of 150
µl. Mix gently by rocking the plate back and forth, then left to right.
Do not move the plate in a circular motion, as this may cause the cells to
preferentially sediment around the edges of each well.
- Incubate cells in an incubator for 16-24 hours.
- After 16-24 hours of transfection, change the medium to complete growth
- Measure Transfection Efficiency by assaying for transfection conditions
that yield greatest signal without significant effects on cell viability
Carry out the luciferase assay using either the Dual-Luciferase Reporter
Assay System or Dual-Glo Luciferase Assay System from Promega. Follow the
manufacturer's protocol for developing the assay. Each Cignal Finder Array
plate comes along with a white self-adhesive sticker, which should be
attached to the bottom of the plate before reading the luciferase
activity. Using the sticker to cover the optical bottom of the 96-well
plate helps to maximize the signal-to-noise ratio of each reading.
Expression of the Monster GFP reporter can be monitored via FACS, flow
cytometry, fluorescent microscopy, or standard fluorometry. The spectral
properties of the Monster Green Fluorescent Protein are slightly
red-shifted compared to other commercially available GFP reporters. We
recommend using the standard FACS settings of an argon laser (488nm
excitation) and filters of 530+15 nm (530/30nm) for emission. When
analyzing GFP expression via fluorescent microscopy or standard
fluorometry, we recommend using standard fluoroisothiocyanate (FITC)
filters [excitation of 470+20nm and an emission filter of 515nm (long
(1) Do not add antibiotics to media during transfection as this causes
(2) Avoid the use of DMEM medium*.
(3) Users should use:
- 4 wells for each transfection condition
- 1 Empty well with transfection reagent alone (text for transfection
reagent toxicity, etc.)
- 1 Empty well with media alone (cell viability control)