Goat anti mouse fitc

Thawed PBMC were permeabilised, fixed with 90% methanol, then stained using antibodies to acetylated (Ac) histone (H)3, and Ac lysine (Millipore, Billerica, MA) and Ac H4 (kind gift from Dr Jeff Lifson, National Cancer Institute Frederick, Frederick, MD) and associated isotype controls with secondary staining with either goat-anti-rat PE or goat anti-mouse-FITC (Invitrogen). Lymphocytes were gated by size and data expressed as MFI above isotype control. Fold changes were determined by comparison of MFI at each time point above baseline MFI.

Cells in 6 well plates were washed twice with PBS and treated with 0.25% trypsin-EDTA (Life Sciences) for 5 minutes to obtain single cell suspensions. Trypsin was inactivated with medium containing 10% FBS. Cells were counted, centrifuged at 300g for 5 min, washed twice with PBS and subsequently fixed in 2% paraformaldehyde (USB). Following 15 min incubation at room temperature cells were washed in PBS and in perm/wash buffer (BD) and resuspended at 4x10⁶ cells/ml in blocking buffer comprising perm/wash buffer with 0.1mg/ml human IgG (Sigma) and 10% serum from the species of secondary antibody (Life Technologies). Cells were incubated for 30min at 4°C and 50μl aliquots (2x10⁵ cells) transferred to individual 5 ml polystyrene round-bottom FACS assay tubes. For double staining of cells for OCT4 and SOX17, cells were incubated in perm/wash buffer with mouse anti-OCT4 (Cell Signaling) for 1h at room temperature, following by two washes with PBS and incubation for 1 hour at 4°C with goat anti-mouse-FITC (Molecular Probes) and goat anti-SOX17-APC (R&D Systems). Samples were washed twice and resuspended in 0.2% FBS in PBS in a final volume of 300μl/tube. Separate staining for OCT4 and SOX17 was performed analogously. Cells were analysed on a FACSCalibur flow cytometer (Becton Dickinson) and data analysed using CellQuest software.

For detection of stage-specific markers, cells were grown and differentiated in 96 well plates (μClear, Greiner). Cells were rinsed twice with PBS, fixed in 4% paraformaldehyde (USB) for 15 min at room temperature (RT) and then washed twice with PBS and blocked for 30 min at RT in 1% BSA (Life Technologies) and 0.1 mg/ml human IgG (Sigma) in perm/wash buffer (BD). Cells were subsequently stained for 2 hours at RT or overnight at 4°C with rabbit anti-OCT4 (Cell Signalling), mouse anti-SOX17 (Abcam) or isotype control antibodies diluted in perm/wash buffer. Cells were washed with perm/wash buffer and incubated at 4°C in the dark with goat anti-mouse-FITC and chicken anti-rabbit-Cy5 (Molecular probes) diluted 1:400 in perm/wash buffer. After 1h of incubation, cells were washed with PBS and incubated with Hoechst 33342 (Life Technologies) for 15 min at room temperature. After final washing with PBS 96 well plates were then imaged with IN Cell Analyzer 2000 (GE Healthcare).

Cultures of GAS were grown to log-phase OD 0.4–0.8 and harvested for 5 min at 4000× g. The bacteria were suspended in PBS with 10% goat serum and incubated for 20 min at RT. A 1-mL suspension per test condition was harvested before incubation in 100-μL staining buffer (0.1% BSA and 10% goat serum PBS) with and without 1:4 dilution of immune mouse serum for 1 h at 4 °C. Cells were washed with 0.1% BSA-PBS before incubation in 100-μL goat-anti-mouse-FITC (Thermo Fisher Scientific, Waltham, MA, USA, A16067) 1:3000 in 0.1% BSA-PBS for 45 min at 4 °C. Cells were washed with 0.1% BSA-PBS and suspended in 1 mL of fixative (2% formaldehyde and 50% PBS). Sample data was collected on a BD FACS Canto II flow cytometer, with further analyses carried out using FlowJo Software.

HEK293(hGHR) cells, 5 × 10⁴ per well, were seeded on poly-lysine-coated eight-well chamber slides (Thermo Fisher Scientific), and transfected with a total of 0.5 μg plasmid DNA. For co-transfections, a 1:1 ratio of plasmid DNA was employed. Following GH treatment (100 ng ml⁻¹, 30 min), cells were washed in cold PBS and fixed with cold methanol, 5 min at −20 °C. For immunofluorescent staining, cells were blocked with 5% goat serum and exposed overnight to primary mouse-anti-FLAG (1:500) and/or rabbit-anti-Myc (1:50) antibodies prior to treatment with secondary goat-anti-mouse-FITC (1:500; 626511; Thermo Fisher Scientific, Carlsbad, CA) and/or goat anti-rabbit-A555 antibodies (1:500; #A-21428; Life Technologies) for 4 h. Hoechst 33342 (Thermo Fisher Scientific) was added (15 min) and coverslips mounted with ProLong Gold (Thermo Fisher Scientific). Fluorescent images were obtained using a wide field deconvolution system (GE Healthcare) consisting of an inverted Nikon TE 200 Eclipse microscope, a Kodak CH350 CCD camera, and the Deltavision operating system. Images were acquired using a ×60 objective in a 1024 × 1024 format and deconvolved with nine iteration using SoftWoRx software (Applied Precision, GE Healthcare).