2 Materials
Use ultrapure water to prepare all necessary buffers/solutions. The
supplemented RPMI medium and ethanol solutions may be
prepared in advance. However, the Diluent buffer and AEC substrate solution is to be prepared on the day of the assay as described
below.
2.1 Solutions
and Reagents
1. R10 medium: RPMI 1640 medium supplemented with 10%
fetal bovine serum, 1% glutamine, 1% penicillin/streptomycin,
and 55 μM 2-mercaptoethanol: Stored at 2–8
C.
2. Dulbecco’s phosphate buffered saline (DPBS) without calcium
and magnesium.
3. Diluent buffer: Dilute fetal bovine serum (FBS) solution
200-fold in DPBS to achieve a 0.5% FBS solution.
4. 3-Amino-9-ethylcarbazole (AEC) substrate set (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA): Stored at 2–8
C.
5. 35% and 70% ethanol.
6. Dimethyl sulfoxide (DMSO).
Fig. 1 Schematic overview of experimental procedures
Quantification of Antigen-Specific T cells
527
Use ultrapure water to prepare all necessary buffers/solutions. The
supplemented RPMI medium and ethanol solutions may be
prepared in advance. However, the Diluent buffer and AEC substrate solution is to be prepared on the day of the assay as described
below.
2.1 Solutions
and Reagents
1. R10 medium: RPMI 1640 medium supplemented with 10%
fetal bovine serum, 1% glutamine, 1% penicillin/streptomycin,
and 55 μM 2-mercaptoethanol: Stored at 2–8
C.
2. Dulbecco’s phosphate buffered saline (DPBS) without calcium
and magnesium.
3. Diluent buffer: Dilute fetal bovine serum (FBS) solution
200-fold in DPBS to achieve a 0.5% FBS solution.
4. 3-Amino-9-ethylcarbazole (AEC) substrate set (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA): Stored at 2–8
C.
5. 35% and 70% ethanol.
6. Dimethyl sulfoxide (DMSO).
Fig. 1 Schematic overview of experimental procedures
Quantification of Antigen-Specific T cells
527