inconsistent/patchy if solution is added or removed from the
membrane at high pressure/speed. This is also why the substrate is washed off by submerging the plates in water instead of
running tap water directly over them.
6. C57Bl/6 mice are routinely used in ovalbumin vaccine/tumor
studies, as the above mentioned epitopes bind strongly to the
MHC haplotypes found in this mouse strain.
7. When removing the spleens, try to carefully trim off any large
pieces of associated fat (whitish mass on spleen) with the surgical scissors prior to placing the spleens in the R10 containing
tube. This will help in the homogenization process. Also, when
homogenizing, darker foci can occasionally be found in a small
number of spleens. They are more difficult to homogenize and
can be discarded once the surrounding reddish tissue is placed
into suspension.
8. If counting with a hemocytometer, please note that the cell
suspension will contain both red and white blood cells
(RBC/WBC). As they are the cells of interest, include only
the white blood cells in your counts. They can be differentiated
based on their size and morphology. Alternatively, if using an
automated cell counter, some are compatible with nucleic acidsensitive dyes such as acridine orange which will positively stain
WBC but not RBC.
9. The cell amount added per well can be reduced based on
availability of cells or strength of signal (i.e., number of spots
is too high leading to saturation of the well). However, the
number of SFCs obtained may not be linear (i.e., if you obtain
~100 spots with 400,000 splenocytes, you should not necessarily expect to obtain ~50 spots with 200,000 cells). As the
number of lymphocytes is reduced, there will also be an impact
on the number of antigen presenting cells and the degree of
cell-to-cell contact. So users must confirm the linearity in the
system if planning to use different cell concentrations. If
responses found to be nonlinear, the level of response cannot
be directly compared between experiments using different
amounts of cells.
10. As conA is non–antigen-specific mitogen, it should stimulate
all samples to some degree allowing the user to confirm that all
samples contain viable cells. Even if a similar number of cells are
stimulated with conA, the number of resulting SFCs may differ
between mouse samples. Again, if the response with the positive control is so strong as to saturate the well, the number of
cells can be reduced for this test condition only. In an effort to
save space on the plate, it may not be necessary to include the
conA control for all samples once the user is confident with the
quality of their samples.
534
Bassel Akache and Michael J. McCluskie
membrane at high pressure/speed. This is also why the substrate is washed off by submerging the plates in water instead of
running tap water directly over them.
6. C57Bl/6 mice are routinely used in ovalbumin vaccine/tumor
studies, as the above mentioned epitopes bind strongly to the
MHC haplotypes found in this mouse strain.
7. When removing the spleens, try to carefully trim off any large
pieces of associated fat (whitish mass on spleen) with the surgical scissors prior to placing the spleens in the R10 containing
tube. This will help in the homogenization process. Also, when
homogenizing, darker foci can occasionally be found in a small
number of spleens. They are more difficult to homogenize and
can be discarded once the surrounding reddish tissue is placed
into suspension.
8. If counting with a hemocytometer, please note that the cell
suspension will contain both red and white blood cells
(RBC/WBC). As they are the cells of interest, include only
the white blood cells in your counts. They can be differentiated
based on their size and morphology. Alternatively, if using an
automated cell counter, some are compatible with nucleic acidsensitive dyes such as acridine orange which will positively stain
WBC but not RBC.
9. The cell amount added per well can be reduced based on
availability of cells or strength of signal (i.e., number of spots
is too high leading to saturation of the well). However, the
number of SFCs obtained may not be linear (i.e., if you obtain
~100 spots with 400,000 splenocytes, you should not necessarily expect to obtain ~50 spots with 200,000 cells). As the
number of lymphocytes is reduced, there will also be an impact
on the number of antigen presenting cells and the degree of
cell-to-cell contact. So users must confirm the linearity in the
system if planning to use different cell concentrations. If
responses found to be nonlinear, the level of response cannot
be directly compared between experiments using different
amounts of cells.
10. As conA is non–antigen-specific mitogen, it should stimulate
all samples to some degree allowing the user to confirm that all
samples contain viable cells. Even if a similar number of cells are
stimulated with conA, the number of resulting SFCs may differ
between mouse samples. Again, if the response with the positive control is so strong as to saturate the well, the number of
cells can be reduced for this test condition only. In an effort to
save space on the plate, it may not be necessary to include the
conA control for all samples once the user is confident with the
quality of their samples.
534
Bassel Akache and Michael J. McCluskie