Tali® Image Cytometer Software Update 2.1
Highlights of Version 2.1 Tali® Instrument Firmware
- Cell cycle analysis
The Tali® Image-Based Cytometer can perform cell cycle analysis using propidium iodide stained cells, and the cell cycle data can be downloaded in an Excel® spreadsheet–compatible or FCS format. The FCS formatted data can be transferred to any flow cytometry modeling software.
- Fine tuning of fluorescent thresholds
In addition to slider-bar adjustments to set thresholds, users now have greater control by using small-increment adjustments and can also enter absolute values for threshold. This new functionality gives the user precise control over threshold values and allows for easier experiment-to-experiment comparison.
- Adjustable x-axis
You can easily adjust the x-axis and expand or zoom into the region of interest. This helps to separate and identify cells exhibiting low fluorescence.
- Auto-alignment capability
We have eliminated manual camera alignment and replaced it with an auto-alignment program, which performs alignment using Tali® fluorescent alignment beads. This minimizes user-to-user variability in camera alignment and improves data consistency.
Tali® Image-Based Cytometer Home Screen 2.1 Home Screen View
We’ve made three key changes that you’ll notice on the Home Screen
- Addition of the Cell Cycle analysis program
- The Viability and Apoptosis analysis programs are now accessed by selecting Cell Health
- The GFP/RFP button has been changed to Green/Red to better reflect the capability of the Tali® Image-Based Cytometer to detect red and green fluorescent dyes in addition to GFP and RFP fluorescent proteins. See a list of the dyes that are compatible with the Tali® instrument.
Cell Cycle Analysis Program
The Cell Cycle analysis program allows users to collect cell cycle data on a population of cells that have been stained with propidium iodide (PI). When the instrument is in Cell Cycle mode, selecting the Data tab reveals a screen similar to that shown below.
The Analysis tab displays thumbnails of the plots of the various data sets for a particular experiment, and selecting the Propidium Iodide thumbnail expands the cell cycle data histogram, as shown below.
In the expanded histogram, three fluorescent threshold bars separating the four cell cycle regions are displayed. The four cell cycle regions identified in the histogram are color coded to match the numeric data on the Analysis tab. In this example Region A (in red) represents cell debris, Region B (in orange) contains cells in G0/G1 phase, Region C (in blue) shows S-phase cells, and Region D (in green) represents cells in G2/M phase.
Fine tuning fluorescence thresholds and x-axis range
For advanced cell cycle analysis, the three fluorescence thresholds can be fine tuned. Tap on any one of the three threshold numbers, and a keypad is displayed. Type in the new value and press OK. The histogram and numeric data are automatically updated to reflect the new value.
Additionally, you can modify the range of the x-axis to zoom in on a region of interest. Tapping the number on the left side of the x-axis displays a keyboard. Type in the new value and press OK. Repeat with the number on the right. The screenshot below shows the histogram that results from altering the experiment above to have the x-axis adjusted to the range of 600–3,000 RFU.
Cell Cycle Staining, Data Acquisition, and Analysis on the Tali®
For cell cycle analysis, single-cell suspensions are fixed in 70% ethanol overnight and then stained with propidium iodide (PI). Minimally, 500 µL of cells at 1 x 106–5 x 106 cells/mL for each experimental condition is needed. The following protocol is valid for both suspension and adherent cells. For suspension cells, begin at Step 1, below. For adherent cells, first trypsinize the cells and resuspend them in excess complete medium to create a single-cell suspension and neutralize the trypsin; then begin at Step 1 below.
- Ice-cold 70% ethanol in dH2O
- DPBS (without–Ca2+,-or –Mg2+) (Cat. No. 14190)
- Triton® X-100
- RNase A (Cat. No. 12091-039, 20 mg/mL stock)
- Propidium iodide (PI) (Cat. No. P3566, 1 mg/mL stock)
- PI staining solution:
- 0.1% Triton® X-100 + 0.2 mg/mL RNase A + 20 µg/mL PI in DPBS
|Triton® X-100||10 µL|
|RNase A||100 µL|
- Transfer cells to a conical tube.
- Wash cells.
- Centrifuge at 500 x g for 5 min.
- Remove medium and resuspend in DPBS.
- Centrifuge at 500 x g for 5 min.
- Transfer tubes to ice.
- Fix with ice-cold 70% ethanol in dH2O.
- Remove DPBS.
- Slowly resuspend cells in ice-cold 70% ethanol to a concentration of 1 x 106 to–5 x 106 cells/mL. To ensure a single-cell suspension of fixed cells, begin the initial addition of 70% ethanol very slowly (dropwise) while gently vortexing. It is very important that cells are fixed into a single cell suspension. Cells can tend to clump during the fixation step. Very slow dropwise addition of the first 1ml of 70% ethanol while gently vortexing will help prevent cells from clumping.
- Place cells at –20°C overnight. These cells can be kept for weeks at –20°C before staining and analysis.
Stain with PI
- Wash cells.
- Centrifuge cells at 1,000 x g for 5 min at 4°C.
- Remove ethanol, and resuspend cells in DPBS. Ethanol-fixed cells can be difficult to pellet, and it can be very difficult to see the cell pellet at this step. To reduce the risk of aspirating the cell pellet, it may be advisable to decant the supernatant (instead of aspirating it) after pelleting the cells. If cell density is very low, you can resuspend cells in DPBS + 1% BSA in this step. The addition of BSA will help pellet the cells.
- Centrifuge cells at 500 x g for 10 min at 4°C.
- Stain with PI
- Remove DPBS and resuspend in PI Staining Solution to 1 x 106 to–5 x 106 cells/mL (We recommend a final volume of 500 µL, but smaller volumes can be used if needed.)
- Incubate at room temperature for 30 min in the dark.
Acquisition and analysis on Tali® Image-Based Cytometer
- Perform the Cell Cycle assay.
- Tap the Cell Cycle button to launch the program, and enter a name for the file. See Figure 1
- Load and run sample. See Figure 2
- Load 25 µL of PI-stained cell sample into the Tali® Slide.
- Insert slide.
- Enter the number of fields to capture. We recommend that you collect 20 fields per sample. Collecting more fields per sample gives more robust data with less noise.
- Focus and run the sample. See Figure 3
- Analyze results.
- Using an untreated sample (one exhibiting a normal cell cycle histogram), gate on cell size. In this example, small cells (corresponding to debris) and large cells (corresponding to aggregates) are gated out of the analysis. See Figure 4
- Next, set threshold gates for each cell cycle phase. This can be achieved in three ways:
- Tap to select one of the three threshold bars (grey vertical bar on histogram). The selected threshold bar will change from grey to blue. Tap on the arrows at the bottom of the histogram to move the threshold bar.
- Tap to select one of the three threshold bars. The selected threshold bar will turn from grey to blue. Drag the threshold bar to the desired location.
- Tap on the numbers under "RFU threshold". A pop-up keyboard will appear, and values can be entered manually See Figure 5 and 6
- In Figure 7 Jurkat cells were treated with increasing concentrations of nocodazole to induce G2 arrest. The RFU threshold was set using the untreated sample and held constant across drug-treated samples. In some cases, the RFU thresholds may need to be slightly adjusted for each sample, since peaks may shift in drug-treated samples.
Analysis can be performed as data are being acquired OR can be performed using the commands in the Data tab after data for all the samples have been acquired. The instructions shown below represent analysis via commands in the Data tab, but the procedure is the same if this is performed as each sample is acquired.
- Export data.
- Raw data (in csv and fcs format), individual images, and the PDF report can be exported to a USB drive. The export feature is located in the Data tab. Analysis using cell cycle modeling software (such as ModFit or MultiCycle) to obtain accurate estimates of the percentage of cells in each phase of the cell cycle is recommended, especially for arrested or non-normal cell cycle histograms.