Wound-healing experiment in Stencell chambers

1)     Using tweezers, discard the windows of a Stencell
2)     Using tweezers, grab the Stencell and place it on your dedicated cell culture substrate (glass coverslip, plastic Petri dish, multiwell plate)
3)     Optional: use a flat surface to make sure the Stencell properly stick to the cell culture substrate.
4)     Immerse the sample in complete cell culture medium and place it in an incubator (37 °C, 5 % CO2).
5)     Detach your cells according to your standard procedure, centrifuge.
6)     Remove the bubbles present at the interface Stencell/cell cuture substrate using a pipette.
7)     Seed the cells and place the sample back in the incubator until cells are spread and fully occupy the windows space.
8)     Carefully remove the Stencell using tweezers while maintaining one corner with a pipette.
9)     Optional: Place the glass coverslip on its dedicated holder.
10)   Image with a microscope at 37 °C, 5 % CO2.

lmmunostaining of cells confined in Stencell chambers

1)  Using tweezers, discard the windows of your Stencell
2)  Using tweezers, grab the Stencell and place it on your dedicated cell culture substrate (glass slide, plastic Petri dish, multiwells plate)
3)  Optional : use a flat surface to make sure the Stencell properly stick to the cell culture substrate
4)  Immerse the sample in complete cell culture medium and place it in an incubator (37 °C, 5 % CO2)
5)  Detach your cells according to your standard procedure, centrifuge.
6)  Remove the bubbles present at the interface Stencell/cell cuture substrate using a pipette.
7)  Seed the cells and place the sample back in the incubator until cells are spread.
8)  Fix with PFA 4 % for 12 min, rinse 3 times with PBS.
9)  Carefully remove the Stencell using tweezers while maintaining one corner with a pipette.
10)  Stain the cells with standard procedures.
11)  Optional: mount the glass coverslip on a glass slide.
12)  Image on a fluorescence microscope.

Key conditions of success

• When you remove the inner elements of your Stencell, use 2 tweezers: 1 to remove and the other 1 to press close to the junction in order to avoid breaking the Stencell. Alternatively, you can cut the junctions with a scalpel.
• When you put the Stencell on top of your culture substrate, softly remove the bubbles and stick the Stencell, patting it with tweezers
• Depending on the substrate hydrophobicity, the droplet might not completely fill the well. You can:
  * Either fill the well with the standard volume. Then, help the droplets stick to the Stencell by connecting the liquid next to the Stencell walls using the pipette tip.
  * Or, you may add an excess volume of liquid (e.g. 30 μL) and remove it afterwards (e.g. 10 μL).
• But: do not overfill (e.g. > 30 μL) the wells, otherwise two droplets may merge.
• When you put your substrate and cells in the incubator. Wait for 1 to 2 hours until cells spread and fully occupy the windows space. If you need to wait longer, be sure  that the droplets do not completely evaporate

Storage conditions 

• The Stencells are packaged between 2 protective foils: if you wish to store them, keep them between these foils, in order to avoid dust deposition.
  

• Storage at room-temperature, stable up to 2 years.

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