First time working with microfluidics? Start with a control experiment
If you are not used to working with microfluidic assays, we recommend that you first start with a control experiment to get used to the set-up. Create a stable gradient and visualise the gradient by adding the fluorescent Gradient Marker product of your choice. In this way you will learn how to work with microfluidic assays, and you will have your own control experiment that shows that the method works as expected.
Microfluidic assays are sensitive to air bubbles – This is how to avoid them!
Overnight temperature equilibration of cell media, and all other solutions you use within microfluidic assays is a key issue and very IMPORTANT. Even the cell media you resuspend your cells in should be equilibrated. Unscrew the caps of the Falcon tubes slightly to allow for gas exchange. Make sure to put the liquids and tubes back into the incubator after having used them for resuspending cells or loading CellDirector outside the incubator in room temperature.
Avoid any bubbles in the pipet tip when pipetting coating solutions and cell suspension into CellDirector.
Work droplet-to-droplet by tilting the assay slightly when injecting solutions or inserting tubes into already filled microfluidic assays. Also make sure you always see excess liquid coming out of the two “inlet connectors” when doing the pre-coating and cell seeding steps.
Load syringes slowly to avoid filling them with air bubbles. Inspect the syringes, as well as the tubes before you connect them to the tilted CellDirector, connect droplet-to-droplet.
Experience air bubbles nevertheless – try this!
Temperature equilibrate all solutions over night as described above. Fill the syringes with the cell media and gradient subtance that suit your experimental set-up. Degass the filled-up syringes by placing them in a degassing chamber until all air bubbles have disappeared. Place the degassed syringes in a 37 °C incubator for temperature and gas equilibration until it is time to start the experiment.
Microfluidic assays contain only small volumes – Avoid evaporation during long incubation times!
The fluid volumes within microfluidic assays are small compared to 96-well plates. Keep CellDirector in a humidified environment to avoid evaporation, especially when working with overnight incubations. When incubating your cells, place CellDirector in a petri dish together with a wet tissue to avoid evaporation.
When for example seeding your cells, make sure to check you do not introduce air bubbles, pipett slowly and check that you can see liquid coming out from both the inlet connectors in CellDirector.
Cells must be able to migrate – Live cell tracking reveals!
Some coatings can make cells adhere so much to the surface that they are not able to migrate for that reason. You discover this easily by studying your time-lapse images. Not all parts of the cell detach when they try to migrate and it can look like they are attached to a rubber band: the cells move towards a direction, but are pulled back.