Seeding HUVECs Onto BioFlux Plate

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Protocol for seeding HUVECs onto BioFlux plates

Coat plate with Fibronectin (Advanced BioMatrix P/N 5080, Human, Lyophilized, 5 mg) Storage: 2 to 10°C prior to reconstitution. Stored at -20°C after reconstitution; avoid repeat thawing and freezing. Use bc 48-wells 0-20dyn plates

  1. Add 5mL of sterile water to yield a concentration of 1ug/uL (10x).
  2. Incubate for 30-60 minutes at 37°C to dissolve. Do not agitate.
    Note: Upon reconstitution, the solution may contain a small amount of insoluble aggregated material. This phenomenon is inherent to fibronectin and does not affect product performance. Reconstituted fibronectin may be stored in aliquots at -20 C. Avoid repeated freeze-thaw
  3. Further dilute this lug/uL stock solution with cell culture medium to achieve the desired working
    concentration of 100ug/mL (i.e. add 100uL of fibronectin to 900uL of media; suffice for 18
  4. Add 50uL diluted fibronectin to inlet wells avoid forming bubbles.
  5. Check for and dislodge any bubbles. Gently tap the plate onto the paper towel covered bench top. Clamp on the interface.
  6. In the Bioflux manual mode, highlight the inlet columns. Apply 10 dyne/cm2 2 for 2 minutes.
  7. Remove the interface and check to make sure there is a small droplet in each OUTLET WELL. This indicates that the fibronectin solution had gone through. Check the channels/view fields for air bubbles
  8. Incubate for 45 to 60 minutes at 15 to 25°C. Note: Prepare HUVEC suspension for seeding during this time.
  9. Rinse off the outlet and inlet wells with 100uL of media. The coated plates should be used immediately and should not be allowed to dry. Proceed immediately to seeding HUVEC.

Seeding plate with HUVEC Perform aseptically and as sterile as possible. HUVECs should already be grown and healthy in culture one week prior to seeding onto plate. Use Detachin (Gelantis P/N T100100) to lift the cells. One to two T75 flasks at 70% confluence should be enough for 18 samples

  1. Place 5uL of HUVEC (30-50 X 105 cells/mL) in the outlet wells. Seed only 2 to 4 wells at a time and within 10 seconds of placing the cells in the outlet. HUVECs adhere to the glass very quickly SO work as fast as possible.
  2. Apply 5 dyne/cm? for to 15 seconds to the outlet wells as needed to perfuse the HUVECs to the channel.
  3. Remove excess media from both inlet and outlet wells. Add 50uL of fresh media to each inlet and outlet wells. Incubate at 37°C 5% CO, for 1 hour
  4. Add 500uL of fresh media to inlet wells. (Optional: Seal with air-permeable plate sealer). Clamp on the interface and perfuse for 10 minutes under 0.2 dyne/cm?. Incubate overnight at 37°C 5% CO2.
  5. The following day: check for formation of HUVEC monolayer. Rinse and remove excess cell growth from the outlet and inlet wells. The HUVEC monolayer can now be used for experiments.

Activation of HUVECS with TNF - a (Thermofisher P/N PHC3015, 10ug)

Positive control = No treatment; to be perfused on activated HUVECs

  1. Briefly centrifuge and reconstitute the vial of TNF-a using 0.2mm-filtered distilled water to a 2000X stock concentration of 0.1ug/uL (i.e. dissolve 10ug lyophilized powder in 100uL of water). Allow the reconstituted solution to sit at room temperature for 5-10 minutes before use. Store as 5 to 10uL aliquots at ≤ -20°C. Avoid repeated freeze/thaw cycles.
  2. Dilute 2uL of 2000X stock TNF-a solution in HUVEC growth medium to a working concentration of 50ng/mL (i.e. to make 4mL, add 2uL of 2000X to 3998uL of media)
  3. Perfuse the 50ng/mL TNF-a onto HUVECs monolayer at 5 dyne/cm? for 30 minutes. Incubate at 5 37°C 5% CO2 for 4 hours.