This video demonstrates a procedure for determining cell viability in cells expressing GFP Using the tally image-based cytometer. GFP transduced cells are stained using the tally viability kit dead cell red, which stains all of the dead cells red with propidium iodide, the cells are pipetted into a tally cellular analysis slide and loaded into the cytometer, which acquires and analyzes brightfield and red and green fluorescence images. Histograms are then generated to display cell count, cell size PI fluorescence intensity, and GFP fluorescence intensity when compared to traditional flow cytometry.
Tali can provide similar data regarding cell viability and expression. However, this image-based cytometry provides additional visual information about the cell population being examined. The main advantage of this technique over existing methods like flow cytometry or fluorescence microscopy is that the tally image-based cytometer provides both quantitative and visual information simultaneously about your cell sample.
In addition, the tally instrument is easier to use, is less expensive and has a smaller footprint than either a flow cytometer or a fluorescence microscope. This allows researchers to quickly perform quantitative assays like viability in GFP expressing cells on the benchtop. In this procedure, U2 OS cells at a concentration of one times 10 to the six cells per milliliter were transduced with nuclear targeted cell light GFP viral construct to sustain dead cells with propidium iodide transfer 100 microliters of the cell suspension to a new micro centrifuge tube.
Note that a concentration of one times 10 to the fifth to one times 10 to the seventh cells per milliliter is recommended for the assay though the concentration does not have to be exact. Next, add one microliter of tally dead cell red reagent. Then vortex briefly to mix, incubate the staining reagent and cell mixture at room temperature in the dark for one to five minutes.
Following the incubation, immediately proceed to analysis on the tally image-based cytometer. The tally uses disposable plastic cellular analysis slides designed specifically for the instrument. Always be sure to hold the slide by the edges to load the slide.
Pipette 25 microliters of sample slowly into one of the half moon shaped sample loading areas. Here, unstained nont transduced control cells are loaded in chamber A and the stained GFP expressing cells are loaded in chamber B.To perform a viability assay in GFP expressing cells, touch G-F-P-R-F-P on the home screen of the cytometer. The assay options will then appear on the right select GFP plus viability.
The instrument will then ask whether the sample series should be named to name the sample series. Press name now. Then using the touch screen type the name of the sample series and press save the tally cytometer will automatically advance to the measure screen.
Next, to measure the background fluorescence, press the background tab on the bottom right half of the measure screen as can be seen here. The default setting indicates that nine fields of cells will be imaged. Now with the control sample facing away from the instrument, insert the slide in the loading port until it stops.
Do not apply force on the background screen touch press to insert new unstained cell control. The slide will automatically be pulled into the instrument and a live image of the cells will be displayed on the screen. Using the focus knob, bring the cells into the proper plane of view while focusing.
Slide the red zoom button to four x or 16 x. To better view the cell population, the cells should be uniformly dark with a bright halo around each. As shown here, cells may be undercounted when the transition between the background and the edges of the cells is fuzzy so that the cell boundaries are not well defined.
Cells may be over counted when they have bright centers and dark perimeters. Once the cells have been brought into focus, touch, press to run unstained cell control. To measure the background fluorescence, the cytometer will automatically capture images and display thumbnails of each field of view.
The progress bar provides an ongoing update of the analysis. After the image capture is complete, the cytometer automatically ejects the slide and provides the data from the analysis of the captured images. Stored data is displayed in the dropdown menu of the background tab.
The background control sample will be assigned the same name as that given to the experiment. To run the PI stained GFP transduced sample, remove the slide from the instrument, flip it around and reinsert it with the transduced sample facing away from the instrument. Press the sample tab and then touch press to insert new sample.
When the image appears on the screen, use the focus knob to bring the cells into focus. As before, then touch press to run sample. After the image capture is complete, the data from the analysis appears under the sample tab and the cytometer automatically ejects the slide appropriately.
Dispose of the tally cellular analysis slide. As biohazardous waste, these slides cannot be reused. Once the sample is run, thresholds can be applied to the sample.
To analyze specific cell populations here, we'll adjust the thresholds to determine the percentage of live and dead cells. In GFP expressing cells under the sample tab, the number and percentage of cells expressing GFP live and dead cells expressing GFP total viability. Average cell size number of cells counted and cell concentration are displayed.
In addition, histogram plots displaying cell size, green fluorescence and red fluorescence are displayed at the bottom of the screen. To set the cell size gate, touch the cell size thumbnail to open the cell size histogram to exclude any debris. Move the blue slider bars to exclude both large and small events.
Touch apply to include the cells within the gates. Next to add the GFP fluorescence to the analysis, touch the GFP histogram thumbnail. To open it.
Move the blue slider bar to set the threshold just to the right of the dimmest peak. Use the control sample as a guide. This allows the analysis to include the GFP positive cells, but exclude autofluorescent cells.
Autofluorescence is frequently observed when biological molecules within cells are excel touch apply to confirm and return to the previous screen. The data displayed under the sample tab should now reflect the gates and threshold set for both fluorescence channels. Next, to separate the PI stain cells from autofluorescence or any background present in the sample, press the PI histogram thumbnail to open it again.
The control sample peak will be displayed as a gray peak on the histogram. The red peak is the sample fluorescence. The background fluorescence is displayed as a peak closest to the zero RFU value on this instrument.
To eliminate the background fluorescence in the PI channel, move the blue slider bar to adjust the threshold. To exclude this peak, use the gray peak from the control sample as a reference. When the threshold in the PI channel is appropriately set, touch apply to confirm and return to the previous screen, the tally instrument will again automatically reanalyze the data and update the results in the sample tab.
Next, to visually confirm that each cell is properly assigned, press the zoom tab, then select a captured field of view for review by pressing the thumbnail of the image. Then press the layers tab. Next, press the GFP or PI button to display the image captured through that channel.
Press the same button again to remove the layer from the display or to superimpose layers, touch buttons corresponding to each channel to identify how the cells are counted through a particular channel. Touch circles the cells that were counted through only the bright field channel, which do not express fluorescence will be circled in blue. This indicates a particular cell is live cells expressing in the green channel.
GFP will be circled in green cells expressing in the red channel. Stained with pi will be circled in red and are dead cells counted in both. The red and green channel will be circled in yellow.
This indicates that the cell expresses GFP but is also stained with pi and therefore is dead. Occasionally a black circle will appear on the screen. These are objects that have been identified but have been excluded from the analysis based on cell size.
Continue to fine tune the threshold on the fluorescence histogram using the steps just described until each cell that is expressing fluorescence is circled with the appropriate color. All analysis parameters are automatically saved to the instrument. Under the data tap.
The tally can store data files from 500 sample runs. Each file stored in the data tab is available for reanalysis. By selecting the file from the data tab, it will be reopened and all histograms and layers become active to archive data and generate reports.
The data stored in the instrument may be transferred to a computer using A USB drive. Four representative cell types were transduced with a nuclear targeted cell light, GFP viral construct stained with tally viability kit using dead cell red reagent and analyzed by the tally in a flow cytometer as shown here. Both methods reported approximately the same percentage of the population for each cell type that were expressing GFP, along with the percent of the total population that were both viable and expressing GFP.
These data demonstrate that the Tali cytometer is capable of discriminating between total GFP expression in a population and GFP expression in the live population. We have just demonstrated how to use the tally image-based cytometer to assess fluorescence expression and viability in your cell sample. This technology bridges the gap between individual and population cell studies.
Using the tally image-based cytometer, you can collect quantitative and qualitative visual information about individual cells as well as larger cell populations. Using the same procedure, several other assays can be performed including apoptosis, RFP expression, and cell viability For non expressing cells, the potential applications include assessment of gene expression to studies of drug efficacy.
