Biology Reference
In-Depth Information
4.
Dulbecco's MEM with 25 m
M
HEPES (Gibco-BRL).
5.
5% (w/v) glutaraldehyde. This solution is hazardous and skin contact should
be avoided.
2.2. For Attachment Assay Only
1.
0.1% (w/v) crystal violet, 200 m
M
MES, pH 6.0. It is important to filter this
solution, as its intense blue color can make it difficult to determine whether it has
dissolved properly. If the solution is not filtered, specks of solid crystal violet can
also be added to the experimental wells, resulting in high absorbance readings.
This solution is hazardous and skin contact should be avoided.
2.
10% (v/v) acetic acid.
2.3. For Spreading Assay Only
Dulbecco's divalent cation-free PBS (Gibco-BRL), 0.05% (w/v) sodium
azide. This solution is hazardous.
3. Methods
3.1. Attachment Assay
1.
Dilute adhesion molecule with Dulbecco's PBS.
2.
Add the diluted adhesion molecule to the wells of the microtiter plate (100
L/well).
Leave a blank well or wells for measuring background spreading on blocked
plastic (
see
Note 2
).
µ
3.
Incubate at room temperature for 60 min or at 4
°
C overnight (
see
Note 4
).
4.
Aspirate, add 200
L 10 mg/mL heat-denatured BSA in divalent cation-free PBS
and incubate at room temperature for 30 min.
µ
5.
While the blocking is underway, prepare a suspension of the cells to be examined
(
see
Note 5
).
6.
Count the cell density on a hemocytometer, resuspend the cells to a concentration
of 5
10
5
/mL for fibroblasts and similarly sized cells and 10
7
/mL for lymphoid
cells in warm DMEM/HEPES (gassed with 5% [v/v] CO
2
), and incubate at 37
×
C
in a 15-mL polypropylene tube for 10 min. For experiments examining the effects
of inhibitory agents, aspirate the BSA, add 50
°
µ
L of 2X agent diluted into PBS
followed by 50
µ
L cells. Add 50
µ
L PBS followed by 50
µ
L cells for control
wells (
see
Note 6
).
7.
The incubation time chosen for attachment assays depends on the cell type, as
some cells adhere more quickly than others, but 15-20 min is usually adequate.
8.
Fix cells in the wells to be used for determining 100% attachment value by addi-
tion of 100
L 5% (w/v) glutaraldehyde. Assays are usually performed in tripli-
cate or quadruplicate.
µ
9.
Remove nonadherent and loosely attached cells by either tapping the plate or
gently washing the wells with one or more 100-
µ
L aliquots of PBS (
see
Note 7-9
).
10.
Aspirate the final wash and fix attached cells by addition of 100
µ
L 5% (w/v)
glutaraldehyde for 20 min at room temperature (or at 4
°
C overnight if necessary).
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