Rat anti mouse cd8β eflour 450

Activation and cytokine expression profiles of infiltrating T cells were investigated by fluorescence-activated cell-sorting (FACS) analysis. 1 × 10⁶ lung T cells were stimulated with RSV-specific peptide (M2_82–90, 1 μM) (Anaspec, Fremont, CA) or PMA/Ionomycin (50 ng/mL and 1 μg/mL, respectively) for 6 h to assess CD8+ T-cell activation and CD4+ T-cell polarization, respectively. Fresh RPMI media without PMA/Ionomycin or M2 peptide was applied to cells as a negative control. Following the stimulation period, brefeldin A (0.5 ng/mL, BD Biosciences) was added to inhibit protein transport for 2 h before extracellular staining with rat anti-mouse CD8β eFlour 450 (clone: eBioH35-17.2) (eBioscience) hamster anti-mouse CD3ε APC-Cy7 (clone: 145-2C11), and rat anti-mouse CD4 PerCP-Cy5.5 (clone: RM4-5) (BD Biosciences). All cells were treated with cytofixation and permeabilization buffer (BD Biosciences) for 20 min prior to staining with rat anti-mouse IL-17A PE (clone: TC11-18H10.1), IFNγ FITC (clone: XMG1.2) and IL-4 APC (clone: 11B11) (BD Biosciences) for 45 min. Cells were washed two times with PBS containing 2% FBS and 0.1% NaN₃ and analyzed on the FACS LSRII (BD Biosciences). At least 10,000 CD8+ events and 30,000 CD4+ events were acquired to ensure sufficient detection of intracellular IFNγ, IL-4, and IL-17A.

Subsets of mice were euthanized at days 5 and 7 post-infection and bronchoalveolar lavage (BAL) fluids collected by washing lungs with 3 × 1-mL saline through a small tracheal incision, previously described (Elliott et al., 2007 (link)). Cell pellets were acquired by centrifugation from lung lavage fluids and stained with antibodies developed against extracellular anti-mouse antigens: hamster anti-mouse CD3ε APC-Cy7 (clone: 145-2C11), rat anti-mouse CD4-PerCPCy5.5 (clone 145-2C110), rat anti-mouse CD45-APC (clone: 30-F11) (BD Biosciences, San Jose, CA) and rat anti-mouse CD8β-eFlour450 (clone: eBioH35-17.2) (eBiosciences, San Diego, CA). Cell data acquisition was completed using the BD Biosciences LSRII. Subsequent gating and analysis were performed using Flowjo software (Asland, OR). In separate experiments, BAL cells were prepared onto cytospin slides at a rate of 5 × 10⁵ cells per slide. Slides were stained with Diff-Quik reagent (Fisher Scientific, Houston, TX) and neutrophils, lymphocytes, and monocyte/macrophage were quantified on at least ten 10X fields per slide.