2.2 Antibodies
and Stimulants
1. Capture antibody to IFN-γ (Clone AN18; Mabtech, Nacka
Strand, Sweden): Stored at 2–8
C (see Note 1).
2. Biotinylated detection antibody to IFN-γ (Clone R4-6A2;
Mabtech, Nacka Strand, Sweden): Stored at 2–8
C.
3. Streptavidin-horseradish peroxidase (HRP) (Mabtech, Nacka
Strand, Sweden): Stored at 2–8
C.
4. Concanavalin A (conA) from jack bean: Dissolved in DPBS at a
concentration of 5 mg/mL and dispensed into single use aliquots, which are then frozen at <À15
C.
5. Synthetic peptides corresponding to specific ovalbumin residues (1) amino acids 257–264: SIINFEKL (CD8 epitope) and
(2) amino acids 323–339: ISQAVHAAHAEINEAGR (CD4
epitope). Peptides are provided as lyophilized powder at
>90% purity and have a trifluoroacetate counterion. They are
dissolved in DMSO at a concentration of 2 mg/mL and dispensed into single use aliquots, which are then frozen at
<À70
C (see Note 2).
2.3 Equipment
and Plasticware
1. Surgical scissors.
2. Forceps.
3. Sterile petri dish.
4. Sterile microscope glass slides with frosted end.
5. Hemocytometer.
6. ELISpot PVDF 96-well microplates, MSIP type (Millipore
Sigma, Burlington, MA, USA).
7. Multichannel pipettes.
8. Reagent reservoirs.
9. 12-Channel reagent reservoirs.
10. P200 and P1000 pipette tips.
11. 15 and 50 mL conical tubes.
12. 70 μm cell strainer.
13. Large plastic container.
14. Orbital shaker.
15. Dissection microscope or automated ELISpot reader.
3 Methods
Steps in Subheadings 3.1–3.4 are to be performed aseptically in a
biological safety cabinet. The procedures described in Subheading
3.5 (plate development) do not need to be performed aseptically.
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Bassel Akache and Michael J. McCluskie
and Stimulants
1. Capture antibody to IFN-γ (Clone AN18; Mabtech, Nacka
Strand, Sweden): Stored at 2–8
C (see Note 1).
2. Biotinylated detection antibody to IFN-γ (Clone R4-6A2;
Mabtech, Nacka Strand, Sweden): Stored at 2–8
C.
3. Streptavidin-horseradish peroxidase (HRP) (Mabtech, Nacka
Strand, Sweden): Stored at 2–8
C.
4. Concanavalin A (conA) from jack bean: Dissolved in DPBS at a
concentration of 5 mg/mL and dispensed into single use aliquots, which are then frozen at <À15
C.
5. Synthetic peptides corresponding to specific ovalbumin residues (1) amino acids 257–264: SIINFEKL (CD8 epitope) and
(2) amino acids 323–339: ISQAVHAAHAEINEAGR (CD4
epitope). Peptides are provided as lyophilized powder at
>90% purity and have a trifluoroacetate counterion. They are
dissolved in DMSO at a concentration of 2 mg/mL and dispensed into single use aliquots, which are then frozen at
<À70
C (see Note 2).
2.3 Equipment
and Plasticware
1. Surgical scissors.
2. Forceps.
3. Sterile petri dish.
4. Sterile microscope glass slides with frosted end.
5. Hemocytometer.
6. ELISpot PVDF 96-well microplates, MSIP type (Millipore
Sigma, Burlington, MA, USA).
7. Multichannel pipettes.
8. Reagent reservoirs.
9. 12-Channel reagent reservoirs.
10. P200 and P1000 pipette tips.
11. 15 and 50 mL conical tubes.
12. 70 μm cell strainer.
13. Large plastic container.
14. Orbital shaker.
15. Dissection microscope or automated ELISpot reader.
3 Methods
Steps in Subheadings 3.1–3.4 are to be performed aseptically in a
biological safety cabinet. The procedures described in Subheading
3.5 (plate development) do not need to be performed aseptically.
528
Bassel Akache and Michael J. McCluskie