LN contains thymus-dependent Tcrγ⁺ NK cells. (A) DNA was extracted from NK cells purified from various tissues of B6 mice. Equivalent amount of NK-cell DNA from each tissue was subjected to genomic PCR for rearranged Tcrγ (Vγ2-Jγ1) gene and separated by agarose gel electrophoresis. Ethidium bromide staining of the agarose gel is shown. (B) Mesenteric (M-LN) and peripheral (P-LN) LN NK cells were purified by cell sorting and DNA extracted. Semiquantitative genomic PCR for rearranged Tcrγ (Vγ2-Jγ1) was performed and analyzed by Southern blot as in Figure 2B. Images from different parts of the same membrane are divided by lines. (C) NK cells from indicated tissues were purified and subjected to semiquantitative genomic PCR for rearranged Tcrγ (Vγ2-Jγ1) gene as in panel B, and the approximate percentages of Tcrγ⁺ NK cells were determined. The results are average of 2 experiments. (D) DNA was extracted from NK cells purified from nude (Foxn1^−/−) mouse LN and subjected to genomic PCR for rearranged Tcrγ (Vγ2-Jγ1) gene as in panel A. Thymocytes from wild-type B6 mice were used as positive control. Genomic PCR for Nkg2a was used as control. Ethidium bromide staining of agarose gel is shown. (E) DNA was extracted from sorted CD127⁺, CD127⁻, and bulk LN NK cells from Tcrβ/Tcrδ-double knockout mice. Semiquantitative genomic PCR for rearranged Tcrγ (Vγ2-Jγ1) gene was performed as in Figure 2B. SYBR Safe (Invitrogen) was used to visualize DNA.