Question

A research had two identical plates of Hela cells. The researcher performed a trypan blue viability test on the first plate. The second plate was transfected with a GFP expression plasmid. Six hours after the transfection the researcher observed the number of fluorescent cells and then performed a trypan blue viability test. The results are show below:

                                                        Plate 1              Plate 2

Trypan Blue Test         Blue                 50                    100

                                    Clear                200                  150

Fluorescence Test        Green              ---                    75       

                                    Clear                ---                    175

Both qualitatively and quantitatively, what can you conclude about the actual procedure of transfecting the cells?

Based upon the cells that are alive after the transfection procedure, what is the overall transfection efficiency of the procedure? (Show your calculations!)

Answer 1

Answer:

Fluorescence Test is the actual test procedure for the transfection:

Trypan blue is to determine whether cells is viable or nonviabe.a viable cell will have a clear cytoplasm whereas a nonviable cell will have a blue cytoplasm is observed with trypan blue.

Green Fluorescent Protein (GFP):

An inherently fluorescent protein often found in marine organisms, such as jellyfish.

Since the discovery of GFP, several other fluorescent reporters have been introduced; e.g. yellow fluorescent protein (YFP).

1. Qualitative analysis - Cells expressing GFP can be visualized directly by fluorescence microscopy. Visual assessment is quick but can be biased depending on the field of view.
2. Quantitative measurement - Flow cytometry enables the measurement of GFP expression in a large population of cells with high sensitivity and represents the most accurate and objective method for determining transfection efficiency.

Trypan blue stains only the dead cell as it cannot pass through the live cells.

1-

If we compare the viability of transfected and untransfected cell we will find there is 20% decrease in the viability in the transfected cells.

Due to transfection, some cells die. It might be due to use of transfection agent, which gives us an idea that the transfection agent not good for cells health.

2) Based upon the cells that are alive after the transfection procedure, what is the overall transfection efficiency of the procedure? (Show your calculations!)

Out of 150 cells which were live after the transfection 75 cell were positive for GFP.

But to calculate we need to take the dead cells also as they were also the part of cells which we transfected

Transfection efficiency = number of cells which were positive for GFP/total number of cells

75/250

30%