Biomedical Engineering Reference
In-Depth Information
surfaces so that the preparation can be moved between an upright and an inverted
microscope without having to be rebuilt.
3.2.5 Preparing Chambers
1. Coverslips should be cleaned to remove oils and contaminants carried over from
manufacturing and handling. We soak the coverslips in dilute Liquinox (Alconox,
White Plains, NY) detergent in warm distilled water and sonicate the beaker containing
them for 10 min. After extensive washing and sonication in distilled water, we soak the
coverslips in 1 M HCl at 50
60
C for 4
6 h. Thereafter, we remove them with forceps
and store them in 100% ethanol until use.
2. At the time of use, individual coverslips are removed with forceps from the ethanol and
blotted on a paper towel. We then briefly pass each coverslip through the flame of an
alcohol lamp to burn off all the ethanol. Then place the coverslip in a 3.5 cm dish (or a
6-well plate) and add cells suspended in culture media. We use media appropriate for
each cell type supplemented with 12.5 mM HEPES buffer, 10% fetal calf serum and
antibiotics (75 U penicillin and 75
ยต
g streptomycin per milliliter; Invitrogen
# 15140-122). It is important to constitutively culture cells with HEPES so that they
remain acclimatized to this buffer.
3. Wipe the aluminum support slide with ethanol and air dry the residual ethanol. Use a
small spatula to apply a thin rim of silicone grease (e.g., high vacuum grease from
DOW Corning, Midland, MI) around the top and bottom margins of the opening. Flame
a cleaned blank coverslip and attach the coverslip to the bottom of the support slide.
Use a pair of forceps to gently tamp the area of the coverslip that overlaps the
aluminum slide. Then, place a few layers of Kimwipes on the coverslip and press the
overlap area gently using fingertips to ensure a tight seal and remove any excess grease.
Place the prepared slide on the filter paper placed in a 10 cm plastic culture dish and
expose it to Ultraviolet (UV) light for 10 min in a tissue culture hood. After putting a
lid on the 10 cm plate to keep the observation chamber sterile, place the plate in an
incubator to equilibrate the chamber to 37
C.
4. Place 3
5 ml of culture media in a small culture dish and keep in a CO
2
incubator for
a few hours before filming begins.
5.
In the tissue culture hood, fill the chamber with
B
0.8 ml of warm, buffered culture
media. Take a coverslip with cells from the culture dish (protocol #2) and quickly place
the coverslip, cell side down, on the chamber. Tamp the coverslip down onto the
silicone grease with forceps, aspirate off excess media, and press using Kimwipes and
fingertips to ensure a tight seal and remove excess grease. Wash the top of the
preparation with water first, and then ethanol using a cotton swab to remove any
residual media that could upon drying leave deposits on the coverslip.
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