Deep UV protocol

Reagents

• Glass coverslips (Marienfeld GmBH)
• PLL(20)-g[3.5]-PEG(2): poly-L-lysine-g-poly (ethyleneglycol), (SurfaceSolutions GmbH, Zurich),
  • Stock solution at 1mg/mL in 10mM Hepes pH 7.4 at 4°C,
  • Use at 0.1mg/mL in 10 mM Hepes pH 7.4;
  • PLL-g-PEG is stable and can be kept for months,
  • It can even be reused, if you need to use a large volume, just sterile filter it before and after use.
• Fibronectin (Sigma, reference F1141), aliquoted in 25 μL aliquots stored at -20°C.
  • Diluted in NaHCO~3 10 mM pH 8.5, stored at +4°C.
• Ethanol 70%
• Optional : Fibrinogen- A488 (green), - A594 (red), -647 (far-red) 1 mg/mL (Invitrogen)

Equipment

• UV ozone oven 185 nm (important: wavelength has to be less than 200 nm) under a hood or equipped with ozone catalyzer(UVO cleaner at the Biomaterials department).
• Synthetic Quartz mask with features (Delta Mask, Toppan, Selba Tech).

Procedure

Glass passivation

1. Wash glass coverslips in Ethanol 70% for 1 minute and dry them with absorbent paper.
2. Illuminate the coverslips with the deep UV lamp for 5 minutes. The distance with the lamp should be 5 cm for the Jelight UVO cleaner.
3. Put a drop of 50 to 200 μl (depending on the coverslip size) of a 0.1 mg/mL PLL-g-PEG solution on a parafilm and put the coverslip on top, activated face in contact with the PEG. Incubate 1 hour (SuSoS say 30 min) at room temperature, in a humid chamber to avoid evaporation.
4. Rinse in ultrapure water and dry in a nitrogen stream (SuSoS recommodation) (original protocol: Wash 2 min in PBS, then rinse 2 times 2 min in H2O)

UV illumination

1. Wear gloves
2. Wash mask with 70% Ethanol (use a soft tissue for cleaning not to scratch the mask). Repeat washing steps if necessary. See also our glass cleaning protocols
3. Put the quartz mask with its metal side (bearing the features) facing the UV lamp
4. Illuminate 5 min to clean the surface and make it hydrophilic.
5. Put 3μl of millipore H2O (for 25 mm coverslip) on the metal side of the mask
6. Place the coverslip with the pegylated side on the water drop.
7. Use a plastic tip to press the coverslip against the mask and remove the excess water Caution: any air bubble will cause enlargement of underlying patterns! This is also true for air bubbles that arise due to evaporation during illumination.
8. Put the quartz mask with its quartz side facing the lamp (coverslips on the opposite side).
9. Illuminate 3 to 10 min Minimal illumination time can vary for different cell types. In our hands, 3 min was sufficient for successful spreading of RPE-1 cells, whereas HeLa cells needed at least 5 min, as they are more sensitive to remaining PEG molecules.
10. After illumination, put 5 mL of water on the coverslip and leave until the coverslip spontaneously floats.
11. Take the coverlip carefully to avoid scratching the mask with the tweezer or use plastic tweezers.
12. Incubate the illuminated surface for 1 h at room temperature with 25 μg/mL of fibronectin solution in 100 mM NaHCO3 (pH 8.5). To visualize the patterns, you can add 5 μg/mL of labeled fibronectin. Use a drop of 100 μl solution on a parafilm. NaHCO3 basic buffer favors protein binding on the illuminated regions.

Planting of the cells

1. Collect cells preferably using EDTA 0,02% in PBS and NOT trypsin. This is particularly important for HeLa cells. Using EDTA allows faster rebinding of the cells in the patterns, which is important to avoid cell clustering before adhering to patterns.
2. Rince EDTA solution with pre-warmed and pH equilibrated culture medium (important for cells to rebind fast).
3. Resuspend cells using a 1 mL micropipette to separate them well.
4. Add 100.000 to 200.000 cells for a 25 mm coverslip or 6-well plate well. Use a small volume (for example 500 μL for a 35 mm well or dish). This will allow cells to fall fast on the surface and to remain homogeneously spread. Leave the dish in the incubator without moving it.
5. Wash unattached cells 20-30 min (depending on the cell type) after plating with equilibrated hot medium. To wash, add medium from one side and aspirate from the other side at the same time to create a gentle ow over cells. Cells should be spread on the patterns 1 hour after plating.

Timing

• PEG coating: 1 hour (coverslips can be stored for a week after that stage)
• Illumination: 10 mins (coverslips can also be stored for a week after that stage)
• Incubation of protein and washing: 1h20 (coverslips should be used in the following 2 or 3 days after that step and kept in PBS at +4°C)
• Plating the cells: 30 min
• Total time: 3 hours

Illumination details for Excimer lamp in Oslo

1. On the nitrogen tank
   1. Make sure that the output valve (blue handle) ius closed
   2. Open the main valve
   3. The two pressure gages show pressure in the tank (left) and pressure out to us (right)
   4. The pressure reduction valve (marked AGA) should be turned clockwise to increase pressure and anti-clokwise to reduce pressure.
   5. The air pistol can be used to let out pressure to get a correct reading on the out pressure
   6. set the out pressure (left pressure gauge) to 2 bar.
   7. open the output vale (blue handle)
2. on top of the UV lamp
   1. read off the pressure of the second pressure reduction valve. It should be at 0.1 bar
   2. with the grey knob reduce or increase the pressure to 0.1 bar.
   3. verify that the electrical switch is set to 0
3. Turn on power supply and if the Voltage (right display) is not already 28V adjust the right hand knobs until it's about 28V
4. Now you can start and stop the illumination with the electrical switch on top of the UV lamp
5. After illumination is finished:
   1. turn off power supply
   2. turn off nitrogen flow (blue handle)
   3. close nitrogen tank (valve on top of tank)

Troubleshooting

The cells are spreading everywhere

This is probably due to bad passivation, the PLL-g-PEG step hasn't been done right or your PEG solution is bad (it could be too old, or the pH might be wrong). Check if the cells don't attach on a coverslip with just PLL-g-PEG coating.

No cells are attaching the surface

The fibronectin might not be attached to the surface. This can be due to insufficient illumination of the PEG with deep UVs. Increase illumination time and/or fibronectin incubation time (or concentration). Also, check your protein solution and buffer (the protein buffer is important and should be at the right pH).

Cells do not spread fully on the patterns

Same as above, increase fibronectin concentration / incubation time/illumination time. Your cells might also prefer another adhesion protein. It is also important, if you want single cells to spread over the entire pattern, to match the pattern size with the size of the cells you use, this can vary a lot. For example HeLa cells like surfaces around 700 μm2 while RPE1 prefer around 900 μm2. Check Fink et al., Lab On Chip, 2007 for more detailed troubleshooting, and also for other patterning techniques. Note that there are many protein patterning techniques and that this one might not be optimal for your cells or application.