Mild food restriction (70% ad lib

Mild food restriction (70% ad lib. obtained from the Genetic Stock Center at the University of South Carolina (Columbia), housed singly in polypropylene cages at room temperature and humidity (22.5 1C and 50% 5% relative humidity), and exposed to either 16L: 8D (long day [LD]) or 8L: 16D (short day [SD]) each day (lights off at 1500 hours EST) for the entire experiment (half of the mice in each photoperiod condition). During an initial acclimation period, all mice Rabbit Polyclonal to PARP (Cleaved-Gly215) were provided AL food (Harlan TekLad 8640; crude protein =22%, crude fat =5%, fiber =4.5%) and filtered chlorinated tap water. Two weeks before the first KLH injection, food intake was measured for four consecutive days for each mouse, and average daily food consumption was calculated. These data were used to establish (restricted) diets to be used later. All procedures were approved by the Ohio State University Institutional Animal Care and Use Committee and comply with current National Institutes of Health guidelines for animal research and Animal Behavior Society guidelines. Experiment Progression Immediately before initial KLH challenge, blood (~100 housed in long-day (LD; (Demas and Nelson 1996). Briefly, 96-well plates were coated with KLH, and then thawed plasma samples were diluted with phosphate-buffered salineCTween (PBS-T; 1:200), vortexed, and added to plates in duplicate. Positive (plasma samples from already determined to have high anti-KLH IgG titers) and negative (plasma from KLH-naive mice) controls were also added to each plate in triplicate. Plates were then sealed, incubated at 37C for 3 h, and washed with PBS-T, and then one of two secondary antibodies (both alkaline-phosphatase conjugated antimouse: IgG1 [ICN Biomedicals, Aurora, OH] and IgG2a [Serotec, Oxford]) was added to each well (dilutions: IgG1, 1:750; IgG2a, 1:2,000); both subclasses were measured in all samples but on different plates. Plates were again incubated (37C for 1 h) and washed with PBS-T, and then each well was treated with for 10 min at 4C), and red blood cells were lysed with buffer (0.16 M NH4Cl, 10 mM KHCO3, and 0.13 mM EDTA). After a second wash, cells were filtered through a 70-=0.45). Neither FR (repeated-measures =0.43) nor photoperiod (repeated-measures =0.79) affected body mass loss before the secondary antigen challenge (Fig. 1). Likewise, neither FR (univariate =0.24) nor photoperiod (univariate =0.43) affected body mass on the day of the second KLH challenge. Effects of FR and Photoperiod on Circulating IgG Mice mounted IgG1 (repeated-measures 0.001) and IgG2a (repeated-measures 0.001) primary antibody responses against KLH because anti-KLH antibody titers increased significantly over time within individuals. However, photoperiod did not affect primary responses (repeated-measures IgG1: =0.39; IgG2a: =0.80). After the primary antibody responses but before the second antigen challenge, IgG1 titer was not affected by FR (univariate =0.85) or photoperiod (univariate =0.85). The same was true for IgG2a; it was not affected by either FR (univariate =0.17) or photoperiod (univariate =0.64). All mice mounted significant IgG1 (univariate =0.02) and IgG2a (univariate 0.001) secondary responses against KLH, as indicated by elevated antibody titers 5 d after the second KLH challenge. To ascertain whether FR affected secondary antibody responses, we examined IgG1 and IgG2a titers 5 d after the secondary antigen challenge as well as the change in titer during this period. IgG1 titers 5 d after the second KLH injections were not influenced by FR (univariate =0.55; Fig. 2=0.84). IgG2a titers 5 d after the second KLH injection were also not influenced by FR (univariate =0.77; Fig. 2=0.77). Similarly, changes in Nexturastat A IgG before the second KLH challenge and 5 d after the second challenge were not affected by treatments. IgG1 and IgG2a produced during Nexturastat A the secondary antibody response were not affected by FR (repeated-measures IgG1: =0.83; Nexturastat A IgG2a: =0.06) or photoperiod.