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. 1997 Mar 3;185(5):909–920. doi: 10.1084/jem.185.5.909

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Precursor CTL (pCTL) frequency analysis. BALB/c mice were immunized with 10⁷ PFU vac control, vac-env, or vac-ss⁻ env. After 21 d, splenocytes from immunized mice were cultured (48 wells/titration) in the presence of 10⁵ naive splenocytes that had been pulsed with the H-2L^d–restricted env peptide for 2 h and then irradiated for 7 d. Culture media contained IL-2, T cell growth factor (from Con A–stimulated rat splenocytes), and methyl-α-d-mannopyranoside. Each well was then split into two equal parts and assayed for env-specific CTL activity against P815 targets pulsed with the H-2L^d–restricted env peptide or with media for 2 h. Positive wells were scored as >3 standard deviations above the percent specific lysis without effectors. The fraction of negative wells is plotted on a log scale versus cell number/well and from this line the pCTL frequency was determined (see Materials and Methods).