6. 1Â phosphate-buffered saline (PBS).
7. 24-well plate.
8. Fibronectin (from bovine plasma, Sigma-Aldrich, Oakville,
ON, Canada).
9. Microcentrifuge tubes (1.5 mL).
10. Pipettes and tips (20 μL, 200 μL, 1000 μL).
11. Glass shell vials with closure (15 Â 45 mm, Kimble Chase,
Rochester, NY, USA).
12. Styrofoam floaters.
13. Dimethyl sulfoxide (DMSO).
14. Hydroxyethyl starch (HES, Bristol-Myers Squibb) or, alternatively, Pentastarch, a 20% w/v HES solution (Preservation
Solutions, Inc., Elkhorn, WI, USA).
15. Chondroitin sulfate A (from bovine trachea, Sigma).
16. Methanol.
17. Liquid nitrogen.
18. Long metal forceps.
19. Fetal bovine serum.
3 Methods
Safety recommendations need to be taken into account (see
Note 2).
3.1 Cell Cultures
on Rinzl Plastic
Coverslips
Cells may be isolated from tissues or purchased. If purchased, the
cells are usually obtained cryopreserved and should be thawed and
cultured according to the manufacturer’s instructions using the
recommended media and reagents for passaging. Cells isolated
from tissue are also typically cultured prior to cryopreservation.
Follow culture confluency and passage recommendations according
to cell type. Ensure that cells have reached appropriate confluency by
examining the cultures under a phase contrast microscope before
trypsinization. Cryopreserve cells when they are at an appropriate
early passage and have the typical morphology of healthy growing
cells. Overgrown and late-passage cells approach senescence and are
not recommended for cryopreservation.
3.1.1 Preparing
Cover Slips
1. Rinzl cover slips come in 18 Â 18 mm configuration and need
to be cut into 9 Â 9 mm squares (use standard dissecting
scissors).
2. In the biological safety cabinet (BSC), transfer the cover slips to
a sterile 100 mm Petri dish using fine-point sterile metal forceps. Pour 70% ethanol, making sure all coverslips are fully
covered. Put lid in place, seal with tape, and leave in the BSC
for at least 30 min before use.
584
Leah A. Marquez-Curtis et al.
7. 24-well plate.
8. Fibronectin (from bovine plasma, Sigma-Aldrich, Oakville,
ON, Canada).
9. Microcentrifuge tubes (1.5 mL).
10. Pipettes and tips (20 μL, 200 μL, 1000 μL).
11. Glass shell vials with closure (15 Â 45 mm, Kimble Chase,
Rochester, NY, USA).
12. Styrofoam floaters.
13. Dimethyl sulfoxide (DMSO).
14. Hydroxyethyl starch (HES, Bristol-Myers Squibb) or, alternatively, Pentastarch, a 20% w/v HES solution (Preservation
Solutions, Inc., Elkhorn, WI, USA).
15. Chondroitin sulfate A (from bovine trachea, Sigma).
16. Methanol.
17. Liquid nitrogen.
18. Long metal forceps.
19. Fetal bovine serum.
3 Methods
Safety recommendations need to be taken into account (see
Note 2).
3.1 Cell Cultures
on Rinzl Plastic
Coverslips
Cells may be isolated from tissues or purchased. If purchased, the
cells are usually obtained cryopreserved and should be thawed and
cultured according to the manufacturer’s instructions using the
recommended media and reagents for passaging. Cells isolated
from tissue are also typically cultured prior to cryopreservation.
Follow culture confluency and passage recommendations according
to cell type. Ensure that cells have reached appropriate confluency by
examining the cultures under a phase contrast microscope before
trypsinization. Cryopreserve cells when they are at an appropriate
early passage and have the typical morphology of healthy growing
cells. Overgrown and late-passage cells approach senescence and are
not recommended for cryopreservation.
3.1.1 Preparing
Cover Slips
1. Rinzl cover slips come in 18 Â 18 mm configuration and need
to be cut into 9 Â 9 mm squares (use standard dissecting
scissors).
2. In the biological safety cabinet (BSC), transfer the cover slips to
a sterile 100 mm Petri dish using fine-point sterile metal forceps. Pour 70% ethanol, making sure all coverslips are fully
covered. Put lid in place, seal with tape, and leave in the BSC
for at least 30 min before use.
584
Leah A. Marquez-Curtis et al.