(c) Methanol is flammable and should be kept away from
open flames or other ignition sources. Keep stock in a
designated flammable solvents storage cabinet. Keep the
methanol bath covered when not in use. Methanol is toxic
if swallowed, adsorbed through the skin, or inhaled. Basic
personal protective equipment (gloves, safety goggles, lab
gown, full-length pants, and closed-toed shoes) is
required. Wash with copious amounts of water in case of
skin contact and move the affected person to a ventilated
area in case of inhalation.
(d) Dimethyl sulfoxide is a combustible liquid which may
cause skin, eye, and respiratory tract irritation. It readily
penetrates the skin and may carry other dissolved materials
into the body. Wear basic personal protective equipment;
wash with copious amounts of water in case of skin
contact.
(e) GelRed
® was used in our membrane integrity assessments
because it was designed specifically to be cell membrane
impermeable and is therefore nontoxic and
non-mutagenic.
(f) Glassware can cause cuts and lacerations when it breaks.
Inspect glassware for cracks before use. Always wear
gloves and safety goggles. Loosen corks to relieve pressure
buildup when transferring glass vials from liquid nitrogen
to the 37
C water bath, and carry out the procedure at
arm’s length, away from the face.
(g) Water baths can get contaminated with bacteria and/or
fungi. Add antibacterial and antifungal drops during routine lab maintenance and always wear gloves.
3. This step should be done in the BSC. The surface area of each
well of the 24-well plate is 2 cm
2 . Thus, if all 24 wells are to be
used, prepare about 12.5 mL (0.5 mL extra) of cell suspension
with a concentration of 40,000 cells/mL and load 500 μL per
well. Therefore, you will need 0.5 Â 10
6 cells to seed all
24 wells containing fibronectin-coated cover slips.
4. The concentrations in this CPA cocktail are double the final
concentration of each component. HES powder as supplied by
Bristol-Myers Squibb and used in our references [11, 12, 19]
has been discontinued. As an alternative, we have validated
Pentastarch (20%, w/v, Preservation Solutions, Inc.) [20].
5. It is best to pick the coverslip by one corner, tilt the glass vial at
an angle, and slide the cover slip in, ensuring that the cell side is
facing up.
Cryopreservation of Endothelial Monolayers
589
open flames or other ignition sources. Keep stock in a
designated flammable solvents storage cabinet. Keep the
methanol bath covered when not in use. Methanol is toxic
if swallowed, adsorbed through the skin, or inhaled. Basic
personal protective equipment (gloves, safety goggles, lab
gown, full-length pants, and closed-toed shoes) is
required. Wash with copious amounts of water in case of
skin contact and move the affected person to a ventilated
area in case of inhalation.
(d) Dimethyl sulfoxide is a combustible liquid which may
cause skin, eye, and respiratory tract irritation. It readily
penetrates the skin and may carry other dissolved materials
into the body. Wear basic personal protective equipment;
wash with copious amounts of water in case of skin
contact.
(e) GelRed
® was used in our membrane integrity assessments
because it was designed specifically to be cell membrane
impermeable and is therefore nontoxic and
non-mutagenic.
(f) Glassware can cause cuts and lacerations when it breaks.
Inspect glassware for cracks before use. Always wear
gloves and safety goggles. Loosen corks to relieve pressure
buildup when transferring glass vials from liquid nitrogen
to the 37
C water bath, and carry out the procedure at
arm’s length, away from the face.
(g) Water baths can get contaminated with bacteria and/or
fungi. Add antibacterial and antifungal drops during routine lab maintenance and always wear gloves.
3. This step should be done in the BSC. The surface area of each
well of the 24-well plate is 2 cm
2 . Thus, if all 24 wells are to be
used, prepare about 12.5 mL (0.5 mL extra) of cell suspension
with a concentration of 40,000 cells/mL and load 500 μL per
well. Therefore, you will need 0.5 Â 10
6 cells to seed all
24 wells containing fibronectin-coated cover slips.
4. The concentrations in this CPA cocktail are double the final
concentration of each component. HES powder as supplied by
Bristol-Myers Squibb and used in our references [11, 12, 19]
has been discontinued. As an alternative, we have validated
Pentastarch (20%, w/v, Preservation Solutions, Inc.) [20].
5. It is best to pick the coverslip by one corner, tilt the glass vial at
an angle, and slide the cover slip in, ensuring that the cell side is
facing up.
Cryopreservation of Endothelial Monolayers
589