Surface patterning

Stenciling Fibronectin on polymer (Ibidi low well 80131)

From Meng 2023.11.16

Stencil patterning

1. The PDMS stamp is inverted and placed onto a hydrophobic uncoated 35 mm μ-dish (ibidi GmbH low well 80131).
   1. PUT AT THE CENTER to avoid NOA touching the edge of the dish.
   2. For PDMS stamps that are not freshly made, use tweezer to press it down with the dish.
2. Deposit 10 ul Norland Optical Adhesive 73 (NOA-73, Norland Inc.) along an edge of the stamp (only the NOA-73 liquid can touch the edge, the pipette tip should not touch and move the stamp), allow NOA-73 to flow through the gaps between the PDMS stamp and dish by capillary action.
3. When the NOA flow-through is completed, the stamp is sealed on all sides using NOA.
4. Cure the NOA stencil under ultraviolet illumination in UV machine (for my machine it takes 3 minutes).
5. Use sharp-end tweezers to separate stamp and cured NOA stencil, then peel off the PDMS stamp (optional: keep the stamp for reuse)
6. Add 100 ul of fibronectin solution (50 μg/ml fibronectin in 1xPBS, add 1/10 volume of AF647-conjugated fibronectin if you want to visualize the shapes), degas until the big air bubbles surface.
7. Incubate at 4°C overnight (can store in fibronectin solution for up to a week)

Cell seeding

1. On the day of cell seeding, remove the fibronectin solution, add 500ul 0.2% pluronics solution (inside and outside the stencil). Use tweezers to remove the stencil from the outer edge.
2. Replace the old pluronics with new 800 ul Pluronic, passivate for 10 min at 37 degrees.
3. Wash thrice with 1xPBS before cell seeding.
4. Number of cells: for RPE1, I seed 5x10^4 cells.
5. Remove unattached cells after 10 mins. Add fresh media.
6. Wait for at least 2 hr before imaging.

Fibronectin on glass

First try

Tested 06.02.2023

Equipment and materials

• Fibronectin (eppendorfs in freezer 1 mg/ml), dilute to 50 ng/ml (? check)
• Fluorescent fibronectin added in ration 1/10
• Matec 35mm petri dishes with glass bottom plasma cleaned (no O2, 100% 5 min). For future: test cleaning and plasma protocol for activation. Petri dishes moved to 431 before stamping. Too long time?
• Plasma cleaning: handheld could not be used because too large distance to glass bottom
• PDMS stamps: cut out from top of patterned silicon wafer. The wafer was broken and maybe some fragments were transferred. Check in WLI. Some messing around because they were turned around. checked in microscope. They are hydrophobic.
• Fibronectin solution drops (about 100 ul) on each stamp. The drops sit on top of the stamp. Pipette tip used to "drag" drop out to the edges. Drops left on stamp for 1h in laf bench under aluminum foil to prevent bleaching.
• Remove drop from stamps with pipette + nitrogen flow
• wash with sterile water (not distilled) and remove with N2 flow (other protocols: PBS)
• Apply stamp to glass, leave for 10 minutes
• Remove stamp (we didn't, but decided it would be best to hold the petri dish up side down to remove the stamp quickly without touching the glass surface again)
• Add BSA solution (powder mixed into PBS 5mg/ml, filter). Leave for 1 h.
• Clean 3 times with PBS, remove with pipette
• Add cells suspended in media.
• We left it only for 10 minutes before removing media with suspended cells and adding new media. The idea was to not have too many cells that did not find patterned areas.