Index
Fibronectin
Attachment Assay
Objective
· Qualitatively assess attachment of HDF cells to different surfaces.
Materials
Non-TC-treated 24-well plates
Fibroblasts cells (HDF)
Fibronectin (Fn)
DMEM (no serum or antibiotics)
PBS
PBS with 10 mg/mL bovine serum albumin (BSA)
Materials as required by cell passaging procedure.
Paint brush
Safety
1. Wear disposable latex gloves at all times
2. Wear safety glasses at all times.
Test Conditions (3 wells / test condition)
A. Control
B. Fn coating entire bottom of well
C. Half Fn / Half control
D. Design wells
1. Dilute fibronectin in DI water to 500 mg/mL. Dissolve by heating in 37 °C waterbath. Do not agitate. Dilute fibronectin to 50 mg/mL with PBS for use.
2. Sterilize paint brushes by soaking in ethanol for 30 min. Dry in laminar flow hood for at least 10 min. Put paint brushes in each hood.
3. Follow cell passaging procedure through step #15.
4. After centrifugation, resuspend cells in 10-15 mL DMEM (no serum or antibiotics). Adjust cell concentration to be near 200,000 cells/mL.
5. Place cell suspension on ice or in water bath until ready to use.
Prepare Protein-Coated Surfaces
1. Aliquot 500 mL of PBS to each of the three control wells (Condition A) on the non-TC-treated 24-well plate.
2. Paint Fn over the surface of half of each of the three wells for the “Half Fn / Half control” (Condition C) on the non-TC-treated 24-well plate. Coat/paint each well 3-5 times to ensure that the Fn completely covers one-half of the bottom of each well. Use liberal amount of Fn; do not be stingy. Also, ensure that the coating is even.
3. Draw a pattern (e.g. alphabetical letter, X, smiley face) with the brush in each of three wells (Condition D) on the non-TC-treated 24-well plate. Again, coat/paint each well 3-5 times to ensure that your design is completely covered with Fn. Use liberal amount of Fn.
4. Aliquot 250-350 mL Fn into each of the three Fn wells (Condition B) on the non-TC-treated 24-well plate. Make sure that the Fn wets (i.e. covers) the entire surface of the well.
5. Place plates in incubator for 30 min.
6. Determine the cell concentration of the batch you are assigned. Do not use more than 0.5 mL of concentrated cell suspension. Test the cell concentration using only one Coulter Counter vial.
7. After the plate has incubated for 30 min, aspirate liquid from wells using glass Pasteur pipet.
8. Rinse each well three times with 1 mL PBS solution containing 10 mg/mL BSA. To rinse cells, do the following:
· Add 1 mL of rinse solution to each well using a micropipette.
· Swish lightly.
· Aspirate rinse solution using glass Pasteur pipet.
· Repeat, as given.
NOTE: When adding rinse solution to each well, squirt the liquid on side wall of well rather than in center of well!
NOTE: Tilt plate slightly to assist in pooling liquid for aspiration. Do not scrape Pasteur pipet tip along bottom of well.
NOTE: To streamline procedure, aspirate all wells, then add rinse solution to all wells, etc.
Seed Cells onto Surfaces
1. Dilute cells to 50,000 cells/mL in DMEM (no serum or antibiotics).
2. Seed 1 mL cells into each test well at 50,000 cells/well. (This seeding density should be near 100% confluency).
NOTE: When seeding, mix the cells gently and often (every 6 wells or every 15 sec) to prevent settling of the cells.
3. Place plates in incubator for 2 hrs.
4. (15 pts) Visually check cell adhesion and cell morphology in wells for Conditions A, B, C, and D using light microscope. Record the pattern of attached cells and extent of spreading. Can you see your design? Describe each half of the “Half Fn / Half control” wells.
5. Aspirate supernatant (media + unattached cells) from each well using glass Pasteur pipet. Do this operation gently to minimize shearing off cells.
6. Add 1 mL of PBS to each well using micropipette. Again, do gently.
NOTE: When adding PBS to each well, squirt the liquid on side wall of well (where there are no attached cells) rather than in center of well (where there are cells)!
7. Aspirate liquid from each well using glass Pasteur pipet. Again, do gently.
8. Add 250 mL of PBS to each well using micropipette. Again, do gently.
9. Check for the presence of unattached cells. If you see a lot of floating cells, repeat steps 5.-8.
10. (15 pts) Visually check cell adhesion and cell morphology in wells for Conditions A, B, C, and D using light microscope. Record the pattern of attached cells and extent of spreading. Can you see your design? Describe each half of the “Half Fn / Half control” wells.
11. Dispose of biohazardous materials properly.
Data and observations should be recorded in your laboratory notebook. The observations recorded in steps 4 and 10 are each worth 15 pts.