3.2 Determination
of Transfection
Efficiency
1. Qualitative evaluation of transfection efficiency is conducted on
an inverted fluorescence microscope (Olympus, Japan). A representative area is selected to visualize the number of cells with
green fluorescence.
2. Quantitative evaluation on GFP transfection is done by counting cells using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
(a) Cells are collected and stained with carboxynaphthofluorescein (CBNF) dye to label live cells.
(b) Cells and buffer solutions are loaded into designated wells
of a Cell Assay Module microchip of Agilent 2100.
(c) The cell Assay Module microchip is inserted into the chip
reader of Agilent 2100. Agilent 2100 Expert Software is
open to run the analysis. At least 500–1500 events are
counted for each sample.
(d) The transfection efficiency of pGFP is calculated as the
ratio of the number of cells emitting fluorescence signal to
the total number of live cells in a sample. The GFP downregulation efficiency by corresponding siRNA probes is
normalized to the control sample with pGFP is delivered
alone.
3. Quantitative evaluation on Luciferase expression is done by
measuring luminescence of cell samples using One-Glo™
Luciferase assay system (Promega, Madison, WI).
(a) Transfer cell samples (post transfected 24 h–72 h) to
96-well plate in 100 μL/well.
(b) Add 100 μL of One-Glo™ reagent to each well and
incubate the plate at room temperature for 10 min.
(c) Measure luminescence with a plate reader (FLUOstar
OPTIMA, BMG LABTECH, Germany). The transfection
efficiency of pLuc is presented as the luminescence of the
total live cells in a sample. The downregulation efficiency
of siRNA is normalized to the control sample with pLuc is
delivered alone.
3.3 Cell Viability
Measurement
1. Transfer 24 h post transfected cells to a 96-well plate in
100 μL/well.
2. Add 20 μL of CellTiter 96 AQueous One solution (Promega,
Madison, WI) to each well and incubate the plate at 37
C for
another 1 h.
3. Measure absorbance at 490 nm with an automated plate reader
(Elx 800, Biotek, VT).
4. Use normal grown cell samples as control and set the viability
to be 100%.
8
Xuan Liu et al.
of Transfection
Efficiency
1. Qualitative evaluation of transfection efficiency is conducted on
an inverted fluorescence microscope (Olympus, Japan). A representative area is selected to visualize the number of cells with
green fluorescence.
2. Quantitative evaluation on GFP transfection is done by counting cells using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
(a) Cells are collected and stained with carboxynaphthofluorescein (CBNF) dye to label live cells.
(b) Cells and buffer solutions are loaded into designated wells
of a Cell Assay Module microchip of Agilent 2100.
(c) The cell Assay Module microchip is inserted into the chip
reader of Agilent 2100. Agilent 2100 Expert Software is
open to run the analysis. At least 500–1500 events are
counted for each sample.
(d) The transfection efficiency of pGFP is calculated as the
ratio of the number of cells emitting fluorescence signal to
the total number of live cells in a sample. The GFP downregulation efficiency by corresponding siRNA probes is
normalized to the control sample with pGFP is delivered
alone.
3. Quantitative evaluation on Luciferase expression is done by
measuring luminescence of cell samples using One-Glo™
Luciferase assay system (Promega, Madison, WI).
(a) Transfer cell samples (post transfected 24 h–72 h) to
96-well plate in 100 μL/well.
(b) Add 100 μL of One-Glo™ reagent to each well and
incubate the plate at room temperature for 10 min.
(c) Measure luminescence with a plate reader (FLUOstar
OPTIMA, BMG LABTECH, Germany). The transfection
efficiency of pLuc is presented as the luminescence of the
total live cells in a sample. The downregulation efficiency
of siRNA is normalized to the control sample with pLuc is
delivered alone.
3.3 Cell Viability
Measurement
1. Transfer 24 h post transfected cells to a 96-well plate in
100 μL/well.
2. Add 20 μL of CellTiter 96 AQueous One solution (Promega,
Madison, WI) to each well and incubate the plate at 37
C for
another 1 h.
3. Measure absorbance at 490 nm with an automated plate reader
(Elx 800, Biotek, VT).
4. Use normal grown cell samples as control and set the viability
to be 100%.
8
Xuan Liu et al.