11. When adding cells, the same tips can be used to add the
samples to multiple wells as long as they do not touch the
wells or stimulant solution already in the plate. To help avoid
contact, position the tips so that they hover above the wells/
plate and eject the solution quickly. It is not an issue to point
the tips toward the bottom of the well in this step as there is
already solution in the well and the tips are not within the well.
12. Ensure the surface in the incubator on which the plates are
placed is flat. Also, do not stack or move plates within the
incubator once placed. The formation of a well-defined
round spot depends on the cell remaining in place during the
entire incubation. If the plates are moved or at a tilt, a cell
producing IFN-γ may shift leading to the formation of a streak
or multiple spots from a single cell. Also, altering the overnight
incubation time may increase/decrease the number of detected
spots, keeping it approximately the same (i.e., ~20 h) throughout a study will help ensure consistency.
13. The initial addition of water causes the cells to swell and burst
through osmosis, making them easier to remove during the
subsequent washes.
14. Tetramethylbenzidine (TMB) can be used in place of AEC as
the substrate in this HRP-based system. TMB is generally more
sensitive than AEC, resulting in more spots, but can also lead to
higher background signals in the media alone wells.
15. Most spots should appear relatively quickly after addition of
substrate. However, the development time with the substrate
can be shortened or lengthened based on the size and number
of spots obtained in your system. Again, to help maintain
consistency, it is important to establish a specific development
time and use it throughout a study.
16. Remove the underdrain with care and pay attention to not
damage or tear the PVDF membranes as they will now be
exposed from the bottom of the plate. A tear in the well can
interfere with the counting of spots.
17. When counting spots, consider that cell-generated spots
should be generally round with a dense center. Fibers, tears,
and debris can lead to the formation of irregular-looking spots,
which should be disregarded when counting.
Acknowledgments
The authors would like to acknowledge Renu Dudani and Blair
Harrison for their technical assistance with the development of this
assay.
Quantification of Antigen-Specific T cells
535
samples to multiple wells as long as they do not touch the
wells or stimulant solution already in the plate. To help avoid
contact, position the tips so that they hover above the wells/
plate and eject the solution quickly. It is not an issue to point
the tips toward the bottom of the well in this step as there is
already solution in the well and the tips are not within the well.
12. Ensure the surface in the incubator on which the plates are
placed is flat. Also, do not stack or move plates within the
incubator once placed. The formation of a well-defined
round spot depends on the cell remaining in place during the
entire incubation. If the plates are moved or at a tilt, a cell
producing IFN-γ may shift leading to the formation of a streak
or multiple spots from a single cell. Also, altering the overnight
incubation time may increase/decrease the number of detected
spots, keeping it approximately the same (i.e., ~20 h) throughout a study will help ensure consistency.
13. The initial addition of water causes the cells to swell and burst
through osmosis, making them easier to remove during the
subsequent washes.
14. Tetramethylbenzidine (TMB) can be used in place of AEC as
the substrate in this HRP-based system. TMB is generally more
sensitive than AEC, resulting in more spots, but can also lead to
higher background signals in the media alone wells.
15. Most spots should appear relatively quickly after addition of
substrate. However, the development time with the substrate
can be shortened or lengthened based on the size and number
of spots obtained in your system. Again, to help maintain
consistency, it is important to establish a specific development
time and use it throughout a study.
16. Remove the underdrain with care and pay attention to not
damage or tear the PVDF membranes as they will now be
exposed from the bottom of the plate. A tear in the well can
interfere with the counting of spots.
17. When counting spots, consider that cell-generated spots
should be generally round with a dense center. Fibers, tears,
and debris can lead to the formation of irregular-looking spots,
which should be disregarded when counting.
Acknowledgments
The authors would like to acknowledge Renu Dudani and Blair
Harrison for their technical assistance with the development of this
assay.
Quantification of Antigen-Specific T cells
535