Biology Reference
In-Depth Information
number of tapping or washing cycles to obtain the best signal:noise ratio (i.e.,
attachment to an adhesive substrate compared to attachment to BSA-blocked plas-
tic). Sometimes this can even be judged by eye in a pilot experiment.
8.
More specific problems include cell death in the assay, which could be caused by
exposure of sensitive cells to heat-denatured BSA, and clumping of cells either in
the centre of a well, or around the perimeter, which is caused by swirling of the
plate. In addition, large errors in attachment assays can result from a) inaccurate
pipeting, which can come from use of multichannel pipetors, or b) suboptimal
washing of wells (note that the volume of BSA blocking solution is higher than
the volume of adhesive substrate, and that the wash step after crystal violet stain-
ing is larger still).
9.
It is advisable to use pipet tips that have their ends cut off for attachment assays.
This is to prevent coated proteins and/or cells being washed off directly by a fine
stream of liquid. Wells should be included to estimate a value for 100% attach-
ment: here, cells are added directly to uncoated plastic or to polylysine-coated
plastic and fixed without washing. The most accurate way of determining this
value is to add cell aliquots corresponding to 20%, 50%, and 100% of the experi-
mental inoculum and then extrapolate the resulting graph. It is also possible that
the absorbance value for 100% may be off the linear range of the plate reader.
Attachment assays rely much more on the accuracy of pipeting than spreading
assays, and therefore it is advisable to use a P200 pipetor rather than a multichan-
nel pipetor wherever possible.
10.
Staining can also be performed overnight without detriment to the final results.
Blank wells should be included to subtract the background binding of crystal
violet to plastic. Avoid getting crystal violet solution on the rims of the wells, as
this dries during incubation and can be difficult to remove by washing.
11.
For cell-spreading assays, an important parameter is the health of the cells. Cul-
tures should be actively growing, but should have been passaged more than 24 h
previously. We have observed relatively poor spreading responses in cells that
were passaged the day before a spreading assay.
12.
Spreading can sometimes be increased by incubating the plate containing the
initial 50-
C for several min to allow them to warm up prior
to addition of cells. To ensure good spacing of cells, guard against swirling,
tapping, or shaking the wells once cells have been added. In our experience, a
single pipeting of cells down the side of the well into the PBS solution produces
good dispersal.
µ
L aliquots at 37
°
13.
Understandably, the optical quality of the plastic that is used to make microtiter
plates is not ideal for phase contrast microscopy. However, the observation of
adherent cells can be greatly improved by adding sufficient PBS/azide to form an
inverted meniscus at the top of the well and then carefully placing a glass cover
slip over the plate. Quantitation of percent spreading is usually carried out by
counting three separate lots of 100 cells selected from random areas of the well.
Both the selection of cells and minimization of double counting are aided by the
use of an eyepiece graticule. Different methods can be used to determine whether
Search WWH ::
Custom Search