Nano-injection has been shown to work nicely with many different cell lines and compounds. Still, whenever a new cell line and/or a new compound is used optimization steps are required.
Generally speaking, these aspects have to be assured:
The cells are viable under working conditions.
The cells stay viable after the mechanical interaction with the FluidFM probe.
The injected solution is not toxic and reaches an effective concentration inside the cell.
Here, we provide the steps needed when designing a new injection protocol.
Cell line handling
The first step is to test if the selected cell line is viable and stable under working conditions. In most cases, we suggest to work with a CO2 independent medium without phenol red, supplemented with FBS and antibiotics. It is important to assure cell viability under these conditions. Therefore, we recommend to perform a 24-hour observation experiment, with a viability test at the end. Adjust the cell handling until its viability is satisfying.
Only once these basic biological requirements are assured injection experiments should be carried out.
Mechanical injection parameters
FluidFM is a very versatile tool and has been already probed to work with several adherent cell lines. However, each cell line shows specific mechanical properties. Therefore, we recommend to determine injection parameters by injecting a harmless compound (e.g. Lucifer Yellow) and measure cell viability after injection.
We suggest the following approach to optimize the mechanical injection parameters:
1. Determine starting parameters based on the online list of already established cell lines. However, we are happy to help you: provide us information regarding your cell line (type, morphology, pictures,…) and we can recommend the bets starting parameters.
2. Perform an injection protocol with Lucifer Yellow, as described here.
3. If the injected cells quickly change morphology (e.g. bubbles or atypical vacuoles) and are highly fluorescent, we suggest to reduce the injection pressure (mbar).
4. If the injected cells die within 1 hour after the injection, we suggest the following actions:
a. Reduce Setpoint value (mV).
b. Reduce Approach Speed (µm/s).
c. Reduce Injection Time (s).
5. Adjust the parameters until injection efficiency and viability are satisfying. Then, apply these parameters for all subsequent experiments. In case a different injected volume is required, injection time (s) can be modified. When working with nanosyringes with small openings, slightly higher pressures (mbar) are allowed.
After determining the optimal mechanical parameters, the cleanliness, concentration and volume of the injected compound must be optimized. For a plasmid or CRISPR complex experiments, where an expression of a protein or a gene is expected, we propose the following approach:
1. Give us a detailed description of the compound (size of the compound, buffer where the compound is diluted, if that compound has been tested by other methods, etc). After analyzing all the provided data, we will suggest you a starting concentration and other pre-treatment advice to assure the compound is not toxic for the cells.
2. Use cell cultures at 50% confluence.
3. Perform the injection of the compound, if possible with a fluorescent molecule to track the process:
a. Assess injection by following the fluorescence of the tracker.
b. Check the expected expression of your compound after the injection:
- If cells show high viability, yet without expression: increase the concentration of the compound, e.g. by factor of 2.
- If cells show low viability: the compound used might disrupt the cell cycle, and therefore, affect the proliferation rate. Also, injecting large volumes into the cell can modify chromatin structure, leading to a decrease in cell viability too. In order to avoid these issues, we suggest to reduce the amount injected into the cell, by diminishing the pressure used and/or the duration of the injection.
As an example, you can follow the template below, where parameters like concentration, injection values, and location of the injection are tested:
c. Repeat and optimize until the desired effect and viability are satisfying.
d. Apply this concentration to a large experimental series to study the biological effect of interest.