Before beginning live-seq experiments, certain properties of the cells that will be experimented on should be understood. These include:
- Estimated RNA content per cell (pg/cell)
- Estimated target gene content per cell
- Well coating
- Baseline cell viability
- Imaging parameters
- Seeding density
- Tracking time interval
Estimated RNA content per cell
When isolating RNA from my cells, how much RNA is expected per cell on average?
An estimation of the RNA concentration per cell can be carried out by isolating a known number of cell and extracting bulk RNA from them.
Estimated target gene content per cell
When sequencing biopsies, how many genes are expected to be detected? Cells with lower target gene content will require larger extraction volumes.
An estimation of the target gene content per cell can be obtained from the literature. Alternatively, for a more accurate estimation, consider carrying out a bulk or single-cell sequencing experiment where possible.
Well Coating
What coating should I use on the base of my well? This is only necessary if the experiment requires the cell to be growing on a specific substrate or if the cells are naturally suspended. Extracts can only be taken from cells that are adhered to a surface, meaning that suspended cells will need to be temporarily attached before beginning the experiment.
Baseline Cell Viability
What is the average viability of the cells in culture? What is the average viability of the cells after a couple of hours in biopsy conditions? Is there a large difference between the two? The biopsy conditions may not be compatible with sensitive cells. The sample is kept at room temperature, as at 37°C, the 1 µL droplets required for the biopsy workflow will evaporate too quickly, and CO2 cannot be used as the doors of the OMNIUM are open so much during the experiment, it would be a danger to the operator. Therefore, it is a good idea to check the viability of non-biopsied cells in these conditions to ensure that there is not excessive death in comparison to normal cell culture conditions.
The baseline cell viability in biopsy conditions is also required to compare it with the viability of biopsied cells. Low viability levels post-biopsy can indicate that the extraction parameters were too harsh.
The baseline cell viability can be measured by simply placing a well of cells in the OMNIUM at room temperature and without CO2 for the expected duration of the experiment. After this time, examine if cell morphology remains similar to that of cells in normal culture conditions and note if there is an increased number of detached cells. To assess viability in this case, a preferred viability method can be used. Having said this, if the cells are to be re-biopsied, a non-endpoint viability assay will be required during the biopsy experiment, as viability is checked before the second biopsy. We recommend sytox green [link: Live-seq/Experimental Design/Feasibility of Biopsy Viability], which can be used with the OMNIUM if you also have the GFP fluorescent filter. However, a different non-endpoint assay of your choosing can also be used. If using a new non-endpoint viability assay on these cells, it is also a good idea to test this out before the biopsy experiment. Check if:
- The assay can distinguish live or dead cells
- The assay results can be seen in the OMNIUM
- The assay does not have any undesirable effects on the cell (i.e. it does not cause death in the cells over extended periods of time)
Imaging parameters
What are the best lighting and magnification settings in the OMNIUM for extracting from cells? This is also of importance for cell tracking if the cell needs to be re-biopsied. As the head will likely be in observation mode during observation, the lighting settings will be slightly different from those required during biopsy.
Define the best objective and lighting required to:
- Distinguish nucleus from cytoplasm, and confidently define the piercing location
- Confidently find and track cells during a timelapse
For example, if cells need to be confluent, in 10x they might seem too small and overlap each other, so 20x would be preferred. Likewise, smaller cells may need a higher magnification to distinguish cells’ features (see Figure 1).
Figure 1. Where is the cell? A) Cell before biopsy; B) Cell before biopsy with outline; C) Cell during biopsy; D) Cell during biopsy with outline. Images were taken using 20x lens, however, a higher magnification may have been clearer
Conversely, far-moving cells might move out of the field of view at a high magnification such as 40x (see Figure 2)
Figure 2. Where did the cell go? Images were taken using a 10x magnification, however, a lower magnification may have allowed the cell to be tracked
If the cell needs to be re-biopsied, a non-endpoint viability assay may require fluorescent staining. These imaging conditions should also be checked:
- Can dead and live cells be distinguished on the OMNIUM?
- What are the best
settings to visualise this?
Seeding density and tracking timing-interval
What is the best seeding density for a live-seq experiment? When tracking cells, how often do they need to be imaged? These are necessary to know if the same cell is to be re-biopsied. If re-biopsy and therefore tracking is not required, cells should simply be seeded at approx. 60-70% confluency for experimentation. For experiments that require re-biopsy, the cell will need to be tracked for the length of time between the first extraction and the second extraction (or the third!) to ensure the same cell is targeted the second time (OMNIUM Manual/Cell Tracking: Cell tracking - FluidFM OMNIUM Manual).
The time interval used between images will depend on the seeding density of the cells as well as the mobility of the cells and how often they split.
- Cells at high confluency (>80%) require lower time intervals (5-15 mins).
- Cells that move around quickly require lower time intervals (5-10 mins). See the small, round cells in Figure 3.
Figure 3. Which cell is the correct one?
Images were taken every 1 minute as cells moved so quickly, it was difficult to
determine which cell went where otherwise.
- Cells that split often (> 2 times per day) require lower time intervals (5-15 mins) or lower seeding densities (60-80%).
To define the time interval required for the monitoring experiment:
- Seed cells in a multi-well plate at the desired cell concentration (or concentrations).
- Place the multi-well plate in the OMNIUM at 37°C and 5% CO2.
- Set the plate to observation mode.
- Select 20 random cells in each seeding density and save them into individual groups per seeding density.
- Set up observation workflow in ARYA using brightfield imaging:
- Action: Record image
- Cantilever position: Retract / Observation height
- Imaging conditions: Defined in imaging parameters
- Focus: Restore focus
- Run the workflow for 2-18 hours.
- Ensure that the UV timer is switched off.
To analyse the results:
- Open tracking tool to determine the latest position of previously selected cells.
- Select the group and the point used during the observation.
- Localise the selected cell in each image:
- Successful tracking:
- Cell is alive and can be tracked through the selected observation time.
- Cell has split and one of the daughter cells can be tracked through the selected observation time.
- The cell has died but has not left the field-of-view.
- Unsuccessful tracking:
- The cell is alive, but cannot be tracked due to too high seeding density.
- The cell has moved out of the field-of-view (alive or dead).
- The cell detaches (alive or dead) and is not trackable until the timepoint of interest.
- Click 'save changes'.
- Close the tool.
- If 16 or more cells can be easily
tracked, these parameters (seeding density and time interval) can be used for a
cell tracking experiment.
If fewer than 16 cells are tracked, consider lowering the seeding density or lowering the time interval between images, and repeat the steps above with the new imaging parameters. - Record the tracking yield for each condition.