14. Count spots using an automated ELISpot plate reader or a
dissection microscope (Fig. 3). Results are routinely reported
as spot forming cells (SFC)/million splenocytes (see Note 17).
4 Notes
1. If interested in measuring the secretion of cytokines other than
IFN-γ, this ELISpot protocol can be adapted through the
replacement of the capture and detection antibodies with
appropriate equivalent antibodies specific to the analyte of
interest.
2. Due to their widespread use, these peptides are available off the
shelf from many different commercial suppliers. If using large
quantities of these peptides, it may be beneficial to place a
custom order with a peptide manufacturer. This usually reduces
the overall cost and allows the user to determine characteristics
such as purity and aliquot size. While the peptides described
here are soluble enough in water to get working solutions, the
solubility of other peptides in water can be low. As such, we
routinely use DMSO as a solvent for our peptides. Solubility is
usually higher, which can be quite useful when working with
other antigens where there is a need to combine multiple
peptides in a pool. However, DMSO can be toxic at higher
concentrations. The final concentration of DMSO when added
to the cells should not exceed 1% (ideally 0.5%). If adapting this
protocol to other antigens where epitopes have not been
defined, cells can be stimulated with whole protein (which
tend to favor activation of CD4 cells [3]) or overlapping peptide libraries which cover the length of the protein (e.g., 15mer
peptides overlapping by 11 amino acids).
3. When washing and coating the plates, it is important not to
allow the wells to dry out after activation with the ethanol or
coating may be uneven. While more expensive, some suppliers
do offer precoated plates where application of coating solution
has been standardized.
4. As live cells will be incubated on the ELISpot plates, it will be
important to take steps to reduce the risk of microbial contamination. While certain steps described above (such as removing
the media by flicking the plate upside down and tapping the
plates on nonsterilized paper towels) may seem risky, they do
not usually introduce any contamination in our experiments.
5. When pipetting into the plates, pay attention to orient the tips
toward the walls of the well instead of straight down at the
membrane. Direct contact with the membranes can introduce
tears. In addition, the background color of the well can be
Quantification of Antigen-Specific T cells
533
dissection microscope (Fig. 3). Results are routinely reported
as spot forming cells (SFC)/million splenocytes (see Note 17).
4 Notes
1. If interested in measuring the secretion of cytokines other than
IFN-γ, this ELISpot protocol can be adapted through the
replacement of the capture and detection antibodies with
appropriate equivalent antibodies specific to the analyte of
interest.
2. Due to their widespread use, these peptides are available off the
shelf from many different commercial suppliers. If using large
quantities of these peptides, it may be beneficial to place a
custom order with a peptide manufacturer. This usually reduces
the overall cost and allows the user to determine characteristics
such as purity and aliquot size. While the peptides described
here are soluble enough in water to get working solutions, the
solubility of other peptides in water can be low. As such, we
routinely use DMSO as a solvent for our peptides. Solubility is
usually higher, which can be quite useful when working with
other antigens where there is a need to combine multiple
peptides in a pool. However, DMSO can be toxic at higher
concentrations. The final concentration of DMSO when added
to the cells should not exceed 1% (ideally 0.5%). If adapting this
protocol to other antigens where epitopes have not been
defined, cells can be stimulated with whole protein (which
tend to favor activation of CD4 cells [3]) or overlapping peptide libraries which cover the length of the protein (e.g., 15mer
peptides overlapping by 11 amino acids).
3. When washing and coating the plates, it is important not to
allow the wells to dry out after activation with the ethanol or
coating may be uneven. While more expensive, some suppliers
do offer precoated plates where application of coating solution
has been standardized.
4. As live cells will be incubated on the ELISpot plates, it will be
important to take steps to reduce the risk of microbial contamination. While certain steps described above (such as removing
the media by flicking the plate upside down and tapping the
plates on nonsterilized paper towels) may seem risky, they do
not usually introduce any contamination in our experiments.
5. When pipetting into the plates, pay attention to orient the tips
toward the walls of the well instead of straight down at the
membrane. Direct contact with the membranes can introduce
tears. In addition, the background color of the well can be
Quantification of Antigen-Specific T cells
533