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Transfection Optimization Reporter Array

Luciferase (Plates)
 
Transfection Optimization Reporter Luciferase Kit: CCA-999L(Plate Format)

The Cignal Finder Transfection Optimization Reporter Array provides a rapid tool for optimizing reverse transfection conditions for future Cignal Finder Reporter Array experiments. The plate format Cignal Finder Reporter Arrays are delivered in a 96-well cell culture plate. The positive control assay is dried down in 8 columns of the plate (8 wells per column). The Cignal Positive Control Assays consists of a mixture of a constitutively expressing firefly luciferase, Renilla luciferase and GFP constructs. Using either a dual-luciferase assay or measuring GFP, you can easily determine the optimum transfection conditions.

Column   Reporter Assay Intended Use
1 Empty Viability & Background Control
2 Empty Viability & Background Control
3 Empty Transfection Reagent Toxicity Control
4 Empty Transfection Reagent Toxicity Control
5 Positive Control Assay Transfection Test Condition
6 Positive Control Assay Transfection Test Condition
7 Positive Control Assay Transfection Test Condition
8 Positive Control Assay Transfection Test Condition
9 Positive Control Assay Transfection Test Condition
10 Positive Control Assay Transfection Test Condition
11 Positive Control Assay Transfection Test Condition
12 Positive Control Assay Transfection Test Condition
 

 

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Cignal Finder Reporter Array (plate format) Specifications

Component Specification Total DNA in each well
Positive control  A mixture of a constitutively expressing GFP construct, constitutively expressing firefly luciferase construct, and constitutively expressing Renilla luciferase construct (20:1:1). 200 ng
 

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  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:
  1. Optimum transfection reagent
  2. Optimum cell density
  3. 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)

  1. 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.
  2. Add varying amounts of transfection reagent in 50 µl of cell culture media per well for each individual transfection.
  3. Follow guidelines of transfection reagent for appropriate incubation times before addition to each well.
  4. 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.
  5. 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 device.
  6. 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.
  7. 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.
  8. Incubate cells in an incubator for 16-24 hours.
  9. After 16-24 hours of transfection, change the medium to complete growth medium.
  10. Measure Transfection Efficiency by assaying for transfection conditions that yield greatest signal without significant effects on cell viability or morphology:

    Dual-Luciferase
    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.

    GFP
    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 pass)].
Notes:
(1) Do not add antibiotics to media during transfection as this causes cell death.
(2) Avoid the use of DMEM medium*.
(3) Users should use:
  1. 4 wells for each transfection condition
  2. 1 Empty well with transfection reagent alone (text for transfection reagent toxicity, etc.)
  3. 1 Empty well with media alone (cell viability control)
 

Kit Components How It Works Manual & Resources Related Products
 
User Manuals Cignal Finder™ 10-Pathway Reporter Arrays (PDF)
White Paper Cignal Reporter Assay Kit: A High Performance Tool for Assessing the Functions of Genes, Biologicals, and Small Molecule Compounds
Web Seminar Attend a live on-line seminar hosted by an Application Scientist about Cignal Reporters
Technical Brochure Cignal Reporter Assay Kits Technical Brochure (PDF)
Power Point Signaling Reporters: Cignal™ Cell-Based Assays for Rapidly Analyzing Signaling Pathway Activity (PDF)
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