Slr 2 flow cytometer

The ability of the ureA transcriptional fusions to drive expression of GFP was assessed visually utilizing an Olympus BX61 fluorescent microscope, as well as using flow cytometry as previously described (Carpenter et al., 2007 (link)). Briefly, strains containing the ureA promoter fusions were grown overnight in liquid cultures containing varying NiSO₄ concentrations (0, 0.5, 1.0, 10 μM) (Sigma). Following overnight growth, 0.5–1.5 ml of each culture was pelleted and resuspended in 1–2 ml of sterile 1× phosphate-buffered saline depending on the density of the culture. Bacterial clumps and culture debris were subsequently removed by passing the resuspended culture through a 1.2-μm Acrodisc PSF syringe filter (Pall). Flow cytometry analysis for the ureA fusion constructs was performed using either a Beckman Coulter Epics XL-MCL flow cytometer with a laser setting of 750 V or a BD SLR II flow cytometer. 20,000 events were collected for each assay. WinList 3D, version 6.0 (Verity Software House) and FlowJo, version X (FLOWJO, LLC) were used to analyze the flow cytometry data. These experiments were performed 3–5 times for each strain-reporter plasmid combination.

Analyses were performed on the green fluorescent protein (GFP)-positive cells 48 hrs after transfections with pCMV-PID1-IRES-eGFP or pCMV-IRES-eGFP control and 24 hrs after drug treatment. AnnexinV/7AAD staining was by flow cytometry using the APC AnnexinV kit (BD Pharmingen catalog #550474) according to manufacturer’s instructions. Mitochondrial depolarization was measured by flow cytometry using the MitoProbe™ DiIC1(5) Assay Kit for Flow Cytometry (Life Technology catalog #M34151). Caspase 3 cleavage staining was assessed using anti-cleaved-caspase 3-pacific blue antibody (Cell Signaling catalog #8788) by flow cytometry. Flow cytometry analysis was performed using an SLR II flow cytometer (BD Biosciences). Cell clumps and sub-cellular debris were excluded using appropriate gating on forward and side light scatter. Data were analyzed using FACSDIVA software (BD Biosciences).

The APC AnnexinV kit (BD Pharmingen cat#550474) was used to assess apoptosis and the APC BrdU Flow Kit (BD Pharmingen cat#552598) was used to assess proliferation, both according to manufacturer’s instructions. Analyses were performed by flow cytometry focusing on the eGFP-expressing or FAM-labeled cells using an SLR II flow cytometer (BD Biosciences).