Facs canto flow cytometry analyzer

Low serum was used to induce G0-arrest of NIH 3T3 cell clones carrying GAPDH BAC transgenes.^{63 (link)} NIH 3T3 cells at 60-70% confluency were maintained in DMEM supplemented with 0.2% BGS (HyClone) for 96 hrs. Cell cycle arrest was assayed by flow cytometry. Cells were detached by trypsinization, pelleted by centrifugation, resuspended in 1XPBS, fixed in ice-cold 70% (v/v) ethanol, and then stored in this 70% ethanol at 4°C. Prior to DNA content analysis, cells were washed in PBS, followed by staining with 2 μg/ml propidium iodide (Sigma) in 1XPBS supplemented with 1 μg/ml DNase-free RNase A (Sigma) at 37°C for 1hour. PI fluorescence staining of DNA was measured on a FACS Canto Flow Cytometry Analyzer (BD Biosciences). Ten thousand events were acquired for each sample. The percentage of diploid cells in G1, S, and G2 was estimated by FACSdiva software (BD Biosciences). FACS analysis of PI stained, serum starved cells revealed >90% of cells with G1 DNA content; the estimated fraction of S phase cells was <4%, in contrast to asynchronously growing control cells which had >15% cells in S-phase.

GFP-ZeoR reporter gene expression levels were analyzed using a FACS Canto Flow Cytometry Analyzer (BD Biosciences) using a 488nm laser to detect GFP and/or Propidium Iodide (PI) through 530/30 or 625/635 nm LP emission filters, respectively. Rainbow fluorescent beads RFP-30-5A (Spherotech Inc.) were used as an intensity standard to normalize GFP fluorescence; the flow cytometer PMT voltage was adjusted to produce the same output intensity for these beads prior to each use of the instrument. Mean cell fluorescence intensity (MFI, in arbitrary units) of GFP normalized to fluorescent bead intensity was used as a measure of transgene expression. To ensure uniform normalization for all samples, fluorescent beads from the same batch were used for all measurements. Untransfected NIH 3T3 and mouse ES cells were used to establish background cell auto-fluorescence levels. Linear fitting of mean GFP expression level versus transgene copy-number for each group of cell clones was performed using Microsoft Excel and fixing the y-intercept “a” to the fluorescence background level of non-transfected cells. The correlation coefficient R² when the y-intercept is fixed is defined as:
$${\mathrm{R}}^2=b{b}^{\prime },\mathrm{where}b=(\sum {\mathrm{x}}_{\mathrm{i}}{\mathrm{y}}_{\mathrm{i}}\mathrm{-a}\sum {\mathrm{x}}_{\mathrm{i}})/\sum {\mathrm{x}}_{\mathrm{i}}^2\mathrm{and}{\mathrm{b}}^{\prime }=(\sum {\mathrm{x}}_{\mathrm{i}}{\mathrm{y}}_{\mathrm{i}}\mathrm{-a}\sum {\mathrm{x}}_{\mathrm{i}})/\sum {({\mathrm{y}}_{\mathrm{i}}-\mathrm{a})}^2.$$