Spleens were removed from naive BALB/c or LPS-resistant C3H/HeJ
mice and pressed through nylon mesh to produce single-cell suspensions.
Red blood cells were lysed with Tris-NH
4Cl. T
cells were subsequently purified by passage through a T-cell enrichment
column (R&D Systems, Minneapolis, MN) according to the manufacturer’s
directions. The enriched, naive T cells (>95% Thy+ cells, measured by
flow cytometry) were suspended in serum-free medium. Serum-free medium
was composed of RPMI 1640 medium, 10 mM HEPES, 0.1 mM nonessential
amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml
streptomycin (all from Bio-Whittaker) and 1×
10
−5 M 2-ME (Sigma) and supplemented with 0.1%
bovine serum albumin (Sigma), ITS+ culture supplement (1 μg/ml
iron-free transferrin, 10 ng/ml linoleic acid, 0.3 ng/ml
Na
2Se, and 0.2 μg/ml
Fe[NO
3]
3; Collaborative
Biomedical Products, Bedford, MA). The proliferation assay used was a
modification of one described previously.
19 To individual
wells of a 96-well V-shape bottomed plate (Corning, Corning, NY), we
added 10 μl 2.5 × 10
4 enriched T cells,
10 μl hamster anti-mouse CD3e IgG (2C11, 2.5 μg/ml; PharMingen, San
Diego, CA), or 10 μl serum-free medium, and 5 μl AqH or PBS. Total
reaction volume was kept constant at 25 μl. The cells were pulsed
with 2.5 μl 20 μCi/ml [
3H]thymidine for the
final 8 hours of the 72-hour incubation (37°C; 5%
CO
2–95% humidified air mixture). On day 3, the
cells were recovered using a cell harvester (model 96; Tomtec, Orange,
CT), and [
3H]thymidine incorporation was
measured in counts per minute, using a liquid scintillation counter
(Betaplate 1205; Wallac, Gaithersburg, MD).