Biology Reference
In-Depth Information
peloruside site, regardless of concentration, the MTs stain as each ligand binds a dif-
ferent site on the MT, and there is no competition between them. If the test ligand is
binding covalently to the taxoid site, no staining of MTs will be seen, whether Flutax
is in excess or not.
Barasoain, D´az, and Andreu (2010)
describe a number of differ-
ent methods for staining and visualizing MTs with fluorescent taxoids, and variations
of these methods may be used to confirm covalent binding.
19.2.7.1
Adherent cell lines
1.
Plate cells onto glass coverslips in culture dishes. Allow the cells to attach
overnight. Cell number can be varied depending on the cell line used. For human
1A9 ovarian carcinoma cells, 1
10
5
cells/dish works well.
2.
Replace the medium with MSA- and/or Flutax-2-containing medium
(25-200 nM) or drug-free (control) medium, and treat cells for 12-16 h.
3.
Incubate with DAPI (10
g/mL) for 30 min at 37
C to stain the nuclei.
4.
Wash twice in PBS and fix the coverslips onto clean glass slides with a small
volume (
m
L) of glycerol buffer (0.13 M glycine/NaOH, 0.2 M NaCl, 70%
glycerol, pH 8.6). Other methods of mounting the slides and fixing the cells
do not work as the glycerol is needed to permeabilize the cells and preserve the
taxoid binding sites.
5.
Fluorescent staining of the MTs can then be examined using a confocal or an
epifluorescence microscope.
10
m
19.2.7.2
Cells in suspension
1.
Seed cells into a 24-well plate (cell number, volume, and plate size can be varied
depending on the cell line being used). For HL-60 promyelocytic leukemic cells,
2
10
5
cells/well are seeded in 500
L.
2.
Treat with MSA and Flutax for 12-16 h, varying the concentrations. For HL-60
cells, a concentration range of 25-200
m
m
M works well with an incubation time
of 16 h.
3.
Add 10
g/mL DAPI and incubate for 30 min, 37
C.
4.
Cytospin 100
m
L from each well onto slides (1265 rpm for 5 min) and mount
coverslips onto slides using glycerol buffer as described earlier.
5.
Examine the fluorescent staining.
m
Alternatively, the cells can be seeded onto plates with coverslips in the wells, and the
plate then centrifuged (400
g
, 5 min) in a cytospin centrifuge to attach the cells to
the coverslips. The coverslips can then be mounted onto slides using a small volume
of glycerol buffer.
To examine staining in the plate directly, cells in suspension can be centrifuged
onto the bottom of the plate. Centrifuge at 400
g
for 5 min, remove the supernatant,
and carefully wash the cells once with PBS and then add glycerol buffer to
cover the surface. Cells can then be examined in an epifluorescence microscope.
For higher-resolution images, examine the cells under a confocal microscope using
glass-bottom plates.
