Harvester 96

The 25-mer peptide used to produce tecemotide (BP25, STAPPAHGVTSAPDTRPAPGSTAPP) was synthesized and purified by CPC Scientific. Two weeks after the final tecemotide booster vaccination, spleens from naïve and vaccinated mice were harvested and prepared as single cell suspensions. CD4+ splenocytes were isolated using a Dynal CD4 cell negative isolation kit (Invitrogen) according to the manufacturer's instructions. These cells (2×10⁵/well) were then co-incubated with irradiated splenocytes (20 Gy, 10⁶/well) from syngeneic naïve mice and 3.1-100 μg/ml of BP25 peptide in flat-bottom 96-well plates. After 4 days, [³H]-thymidine (1μCi/well) was added to each well and incubated overnight. The cells were harvested onto glass fiber filtermats using a Tomtec Harvester 96, and ³H incorporation was measured by liquid scintillation counting on a 1450 MicroBeta counter (Perkin-Elmer). Each sample was run in triplicate, and results were combined to yield a mean ± SE.

Approximately 16 h after ²¹³Bi-irradiation, cells were plated in quadruplicates at 4 × 10⁵ cells/mL in 100 μL in 96-well flat-bottom microtiter plates and incubated at 37°C. Forty-two hours after irradiation, 10 μL of ³H-thymidine (925 kBq/mL; Perkin Elmer) was added to each well and incubated at 37°C. Six hours later (i.e., 48 h after irradiation), cells were harvested (Harvester 96 – Tomtec) on glass fiber Filtermat A (Perkin Elmer). Radioactive emission was amplified with Betaplate Scint (Perkin Elmer) and read using 1450 Microbeta Plus counter (Wallac). Results are expressed as quadruplicate mean ± SD.