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Quantitative Measurement of GFP Transfection Rates in 60 Seconds

GFP
Cellometer Vision
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Introduction

Brightfield Image of HEK293 Cells
Brightfield Image of HEK293 Cells

Fluorescent image of HEK293 cells
Fluorescent image of HEK293
cells. White spots indicate GFP
positive cells.
Cellometer Vision incorporates image based cell counting and fluorescence detection in a compact and easy-to-use instrument. Cellometer Vision is an economical and faster alternative to flow cytometry for many cell population characterization assays such as rapidly determining GFP transfection rates.

Green fluorescence protein (GFP) has been widely applied in cell based assays. The fluorescence signals are used as a readout to monitor gene functions in cell proliferation, differentiations, toxicity, motility, morphology, etc. Currently, to quantitatively analyze fluorescent cells, researchers have to utilize a calibrated flow cytometer. However, in addition to the complexity, cost and availability limitations, flow cytometry is often not the best solution for cell population analysis due to the potential of complex samples clogging the flow system. Time and materials required for analysis can also be inhibitive for rapid and routine cell characterizations such as stem cell research and clinical sample analysis.

Cellometer Vision addresses the need for simple, rapid fluorescence cell counting. By capturing both brightfield and fluorescent images from the same cell sample, Vision can determine the total cell concentration and analyze how many of those cells are expressing GFP. Therefore, using only 20µl of cell sample, researchers can now rapidly identify fluorescence positive cells from a sample, analyze individual cell fluorescence intensity, calculate cell concentration, size and determine the GFP transfection rate in minutes on the lab bench.

Counting results box
Counting results box displays fluorescence cell count, percentage of GFP positive
cells, mean size & concentration.
Cell images and all analysis data, including cell size distribution histograms, can be saved for documentation. Data can also be easily exported to Microsoft Excel spreadsheets for further analysis.

Method

Running Assay:
  1. Load 20µl of sample into the disposable counting chamber
  2. Insert chamber into Cellometer Vision
  3. Select Assay from drop-down menu
  4. Enter Sample ID for this sample
  5. Preview cell images and click 'Count' to begin analyzing sample
  6. Review images and counting data.
  7. Save or export images and/or report data.

Results

Total counted HEK293 cells are indicated by green circles in the brightfield image (Figure 1). GFP (fluorescent) positive, green circled cells can be seen in the fluorescence image (Figure 2). Total and fluorescence positive cells counted, concentration, mean size, and GFP positive percentage are displayed in the results box (Figure 3).

Total counted cells
Figure 1. Total counted cells are
indicated in the brightfield image
by green circles 		GFP positive cells
Figure 2. GFP positive cells are
indicated in the fluorescent
image by green circles

Results
Figure 3. Total concentration (Brightfield), GFP
positive cell concentration (Fluorescence) and
transfection rate percentage are displayed
immediately after image analysis


Cell size distribution histograms (Figure 4), fluorescent intensity vs. size scatter plots and data files can be instantly generated. All experimental data is easily exported to an Excel spreadsheet or saved in a data table.

Cell size histograms are generated instantly
Figure 4. Cell size histograms are generated instantly
Comparison of GFP negative control sample and GFP
Figure 5. Comparison of GFP negative control sample and GFP positive sample.

Cellometer Vision can also be used to detect RFP or YFP transfection rates in a similar manner.

Sample
GFP + Rate
Cell Concentration
GFP+ Concentration
HCT116
58.6%
9.3 x 105 /mL
5.4 x 105/mL
Hela
71.9%
7.0 x 105
5.0 x 105
K562-1
50.5%
7.0 x 105
3.5 x 105
K562-2
70.0%
4.6 x 105
3.3 x 105
HEK293-1
23.4%
5.0 x 105
1.6 x 105
HEK293-2
4.3%
6.3 x 106
2.7 x 105
HEL293-3
25.7%
4.4 x 106
1.1 x 106
HEK293-4
20.2%
7.0 x 105
1.4 x 105
6020
21.1%
4.9 x 105
1.0 x 105
LNCAP
12.1%
3.9 x 106
4.7 x 105
A20
13.5%
5.1 x 106
6.9 x 105
ES (YFP)
95.2%
3.3 x 105
3.1 x 105
CHO (EGFP)-1
1%
4.6 x 106
4.7 x 104
CHO (EGFP)-2
42%
5.5 x 105
2.3 x 105
RPE
31.3%
1.1 x 106
3.5 x 105
Figure 6. GFP Transfection rates of various samples run on Cellometer Vision.


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