Cyan adp high performance flow cytometer

Exponentially growing cells (2×10⁶cells/mL) were seeded in 6 well plates and incubated at 37 °C in 5 % CO₂ overnight. Cells were treated with SG2000 (GI₅₀ dose) for 1 h and subsequently incubated for up to 72 h in drug free media; untreated cells were used as controls. SG2000-treated cells and corresponding untreated controls were harvested by trypsinisation at specific times post-incubation. Cells were pelleted at 200 g for 15 min at 4 °C, washed with cold phosphate buffered saline (PBS), centrifuged and fixed with ice cold 70 % ethanol added drop-wise (samples could be kept at -20 °C for up to 1 week).
Ethanol was removed from fixed samples by centrifugation at 200 g for 5 min at 4 °C and cells were re-suspended in 400 μL of propidium iodide (PI) staining solution (0.05 mg/ml of PI, 0.5 mg/ml of RNAse A and PBS, to a volume of 5 mL). Samples were incubated for 45 min at 37 °C in the dark and processed on a CyAn ADP High-Performance Flow Cytometer (Beckman Coulter, High Wycombe, UK). Gates were drawn to quantify single cells and eliminate debris and doublets (clumped cells). All reagents were obtained from Sigma-Aldrich Co. unless stated.
Analysis of the red fluorescence from PI nuclear staining was performed using Summit 4.3 software (Dako Colorado Inc. Colorado, USA) to quantify the percentage of cells in each phase of the cell cycle.

The phagocytosis assay was performed as previously described with modifications [33 ]. Treated mWBCs (3 × 10⁵ cells) were mixed with opsonized fluorescently labeled transient S. uberis (MOI of 10) in duplicate in a 96-well plate. Labeling of persistent S. uberis strains was not successful (see discussion). The cell mixture was centrifuged at 1200 rpm for 3 min and incubated at 37°C and 5% CO₂ for 45 min. Data acquisitions (10,000 events) were performed by a CyAn ADP High-Performance Flow Cytometer (Beckman Coulter) with red laser (638 nm). Data were analyzed by FlowJo 10 (Treestar, Ashland, OR, USA) [36 (link)].

CD4+ T cells isolated from rhesus macaque or owl monkey whole blood (as described above) or human T cells (Hut78 cells) were analyzed for CD4 expression by flow cytometry (Beckman Coulter CyAn ADP High-Performance Flow Cytometer). Cells were first incubated in Fc receptor (Cd16 monoclonal antibody (3G8), eBioscience Cat #16-0166-82) blocking buffer (PBS + 2% FCS + 1mM EDTA + 0.5% BSA) on ice for 1 hour. These cells were then fixed in 1% PFA and stained for cell surface expression of CD4 (BD CD4 mouse anti-human, APC, clone: L200). On the cytometer, only live cells were gated and histograms of CD4 expression determined by unstained and isotype (BD Mouse IgG₁, Κ, Cat #555751) controls.