In vitro-generated and FACS-sorted Gr-1⁺ mouse granulocytes do not express transcripts for Gzma, Gzmb, and Prf1. (A) mRNA was isolated from in vitro-generated and FACS-enriched (Gr-1⁺) unstimulated or LPS-sensitized (1 μg/mL; 8 hours) B6 and gzmB^-/- granulocytes. Gzma, Gzmb, Prf1, or Hprt1 transcripts were analyzed with specific primer pairs for Gzma, Gzmb, Prf1, and Hprt1 by RT-PCR. As positive and negative controls, 1.3E6SN (lanes 6,8) and EL4.F15 (lanes 7,9) were used. Size of amplified fragments was as follows: Gzma, 291 bp; Gzmb, 135 bp; Prf1, 380 bp; and Hprt1, 249 bp. (B) mRNA was isolated from in vitro-generated unstimulated or LPS-sensitized (1 μg/mL; 8 hours) (lanes 2 and 6,7, respectively) or in addition FACS-enriched (Gr-1⁺; lanes 1 and 5) B6, and gzmA^-/- granulocytes and analyzed as described in “Probing for mRNA transcription,” with specific primer pairs for Gzma, Gzmb, Prf1, Ctsg, and Hprt1 by RT-PCR. Size of amplified Ctsg fragments was as follows: 272 bp; as positive and negative controls, 1.3E6SN (lanes 8, 10), and EL4.F15 (lanes 9, 11) were used.