Johan Dahlin
a522b8c45d
2.6 KiB
MATERIALS AND METHODS
Samples.
Peripheral blood from 197 females was divided into three age categories.
- 23 were cord bloods taken at the time of delivery;
- 94 were from young adults, age range 17– 50 years, median 30 years;
- 80 were from elderly adults > 75 years, range 75–96 years, median 81 years.
All were haematologically normal, with normal full blood counts and white cell differentials for their age. All samples except 63 from young adults were separated into neutrophils and mononuclear cells using standard density gradient centri- fugation (Lymphoprep from Nycomed, Oslo, Norway).
T lymphocytes were purified from the mononuclear fraction using three different methods according to the number of cells available, either E-rosetting using aminoethyl-isothio- uronium bromide hydrobromide treated sheep erythrocytes (Chanarin, 1989) (n¼19), removal of monocytes using carbonyl iron ingestion (Rozenszajn et al, 1984) (n¼38) or CD3-coated magnetic beads from DYNAL (New Ferry, U.K.) (n¼77). Sample purity was assessed morphologically and was >75% (neutrophils range 75–100%, median 95%; lymphocytes range 75–100%, median 97%). High molecu- lar weight DNA was prepared by proteinase K/detergent digestion with phenol/chloroform extraction and ethanol precipitation (Sambrook et al, 1989; Gustincich et al, 1991). X-chromosome inactivation patterns. DNA was screened for heterozygosity of the PGK, HPRT and DXS255 loci and XCIPs analysed in informative individuals using Southern blotting as previously described (Gale et al, 1991, 1992; Gale & Linch, 1994). Analysis of the HUMARA gene was carried out using a radioactive PCR-based technique (Gale et al, 1996). Auto- radiographic signals were quantified using a Hoefer scanning densitometer (Hoefer Scientific Instruments, San Francisco, Calif.) and results are reported as the mean percentage expression of the lower allele from two or more analyses. T-cell receptor g chain gene rearrangement. The g chain of the T-cell receptor gene (TCRg) was amplified using the method of Diss et al (1995). Reaction mixtures (50 ml) contained approximately 100 ng DNA, 1 × Taq buffer from Promega Ltd (Southampton) (10 mM Tris HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100), 1.5 m M MgCl 2, 200 mM dNTPs and 250 ng of each primer. Each sample was analysed using two sets of primers, Vg I þ Vg III/IV with either Jg 1/2 or JPg 1/2. After denaturation at 958C for 5 min, 0.5 unit Taq polymerase was added and 40 cycles performed, each 1 min at 958C, 1 min 558C, 1 min 728C, with a final 5 min extension time at 728C. Products were electrophoresed through 10% polyacrylamide, stained with ethidium bromide and visualized under UV light.