cDNA was amplified using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) Hydroxychloroquine manufacturer with validated gene-specific assays (Applied Biosystems) for CCL11 (eotaxin-1), CCL24 (eotaxin-2), and β-actin on an Applied Biosystems 7500 Fast Real-Time PCR System. RNA expression was reported relative to messenger RNA (mRNA) expression of β-actin for each sample. Serum protein levels of CCL11 and CCL24 were quantified using CCL11 and CCL24 DuoSet ELISA kits (R&D Systems, Minneapolis, MN) following the manufacturer’s protocols. Eosinophils were depleted by pretreating female Balb/cJ mice with 25 μg of sodium azide-free and low endotoxin-tested Siglec-F mAb (E50-240, BD Pharmingen) or isotype
control (Rat IgG2a,κ, R35-95, BD Pharmingen) intraperitoneally in 100 μL of sterile PBS, 24 hours prior to halothane treatment. Since the depleting antibody (anti-Siglec-F) was the same clone as the antibody used to detect eosinophils (PE-anti-Siglec-F), it was anticipated that the mean fluorescent intensity (MFI) of PE on Siglec-F+ cells from the livers of anti-Siglec-F-pretreated mice would decrease in part without the cells being depleted due to competitive binding. To ensure the magnitude of anti-Siglec-F depletion
was CHIR-99021 price not overestimated by flow cytometry, all CD11c− CD11b+ Gr-1low Siglec-F+ and CD11c− CD11b+ Gr-1high Siglec-Flow/neg cells were back-gated to forward- and sidescatter area plots to demonstrate similar granularity and size as the eosinophils and neutrophils isolated from isotype-treated
mice. Similarly, neutrophils were depleted by pretreating female Balb/cJ mice with 10, 20, 25, or 50 μg of sodium azide-free and low endotoxin-tested Gr-1 antibody (RB6-8C5, Bio X Cell, West Lebanon, NH) or rat IgG2b Morin Hydrate isotype control (LTF-2, Bio X Cell) intraperitoneally in 100 μL of sterile PBS, 24 hours prior to halothane treatment. Hepatic eosinophils and neutrophils were quantified by flow cytometry as outlined above. Liver homogenates were prepared and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblot analysis was performed as described,21 except that TFA31 and β-tubulin (Clone AA2, EMD Millipore, Billerica, MA) antibodies were used at 1/2,000 and 1/5,000 dilutions, respectively. Detection of mouse MBP in fixed tissue sections was performed using the established method with rat antimouse MBP (MT-14.7, provided by Drs. Nancy and James Lee at Mayo Clinic Arizona, Scottsdale, AZ) or Rat IgG1,κ isotype control (ab18407, Abcam, Cambridge, MA),32 with some modifications (see Supporting Material for details). All data presented are reported as mean ± standard error of the mean (SEM). Statistical significance between two groups was determined by two-tailed Student’s t test, while statistical differences between multiple groups were determined by one-way analysis of variance with Newman-Keuls post-test analysis. Differences were considered significant when P < 0.05.