Quinacrine

Quinacrine (Sigma) staining was performed as previously described^{39 (link)}. Briefly, 2 × 10⁶ log-phase or purified aged cells were washed once in YEPD + 100 mM HEPES, pH 7.6 and resuspended in 100 μl of the same buffered media containing 200 μM quinacrine. Cells were incubated for 10 min at 30°C and then 5 min on ice. Cells were pelleted and washed twice with ice cold 100 mM HEPES, pH 7.6 + 2% glucose. Cells were resuspended in 100 mM HEPES, pH 7.6 + 2% glucose for imaging. Prior to imaging, cells were kept on ice and all images were obtained within 30 min of staining.
For staining with 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate^{11 (link)} (CDCFDA) (Invitrogen) or 2′,7′-bis (carboxyethyl)-5(6)-carboxyfluorescein^{40 (link)} (BCECF-AM) (Invitrogen), 2 × 10⁶ log-phase or purified aged cells were washed once in YEPD + 100 mM HEPES, pH 7.6 and resuspended in 100 μl of the same buffered media containing 10 μM CDCFDA or 50 μM BCECF. Cells were incubated for 30 min at 30°C and then washed twice with RT 100 mM HEPES, pH 7.6 + 2% glucose. Cells were resuspended in 100 mM HEPES, pH 7.6 + 2% glucose for imaging.
3,3′-dihexyloxacarbocyanine iodide (DiOC₆) (Invitrogen) staining was carried out according to manufacturer’s instructions. Briefly, 2 × 10⁶ log-phase or purified aged cells were washed once in 10 mM HEPES, pH 7.6 + 5% glucose and resuspended in 1 ml of the same buffer containing 175 nM DiOC₆. Cells were incubated for 15 min at RT and then washed twice with 10 mM HEPES, pH 7.6 + 5% glucose. Cells were resuspended in 10 mM HEPES, pH 7.6 + 5% glucose for imaging. Pho8-SEP imaging was also carried out in DiOC₆ imaging buffer after incubation of cells for 20 minutes in the buffer. In all live cell experiments, calcofluor (Sigma) staining of bud scars for age determination was carried out by including 5 μg/ml calcofluor in the first post-staining wash step prior to imaging.
For fluorescence microscopy analysis, cells were visualized under 60X oil magnification using a Nikon Eclipse E800 with the appropriate filter set: UV-2E/C DAPI for calcofluor; FITC-HYQ for GFP, SEP, quinacrine, CDCFDA, BCECF, and DiOC₆; and G-2E/C TRITC for mCherry. Images were acquired with a CoolSNAP HQ² CCD camera (Photometrics) and quantified and processed using Metamorph version 7.1.1.0 imaging software. Cells that exhibited at least four-fold reduction in mean fluorescence intensity, compared to young, wild type cells, were scored as “reduced” in all figures.

A priori, the authors decided to use as potential algorithm components the number of counts of the SLE ICD-9 code (710.0), keyword of “lupus” in the problem list, positive ANA, and ever use of medications frequently used in SLE such as antimalarials, systemic corticosteroids, and disease-modifying antirheumatic drugs (DMARDs) (see below). These components were selected based upon SLE disease criteria and management combined with data accessible in the EHR. Subjects who were not classified as true SLE cases frequently had other autoimmune diseases with ICD-9 codes for these diseases. Multiple true SLE cases had an overlap syndrome (e.g. secondary Sjögren's syndrome or RA). We examined the exclusion of individuals with ICD-9 codes for other autoimmune diseases to assess whether potential SLE subjects who were not true SLE cases were appropriately excluded and also to ensure true SLE cases with overlap syndromes were not excluded. These exclusion criteria were selected during chart review of the training set.
Positive predictive values (PPVs) and sensitivity were calculated for every combination of ≥ 1, 2, 3, and 4 counts of the SLE ICD-9 code; a positive ANA, (titer ≥ 1:40 and titer ≥ 1:160); ever use of antimalarials, systemic corticosteroids, and disease-modifying antirheumatic drugs (DMARDs); and a keyword of “lupus” in the problem list using “and” or “or” between the criteria. PPVs were also calculated excluding ICD-9 codes for systemic sclerosis (SSc) (710.1) and dermatomyositis (DM) (710.3). All algorithms included individuals with at least one count of the SLE ICD-9 code. The PPV was calculated as the number of subjects who fit the algorithm and were confirmed cases on chart review divided by the total number of subjects who fit the algorithm. Sensitivity was calculated as the number of subjects who fit the algorithm and were confirmed cases on chart review divided by total number of confirmed cases. To “fit the algorithm,” the subject had to have available data for that particular algorithm's criteria. If an ANA was not checked at Vanderbilt, it was considered missing. The F-score, which is the harmonic mean of the PPV and sensitivity ([2 × PPV × sensitivity] / [PPV + sensitivity]), was calculated for all algorithms. The F-score is commonly used in informatics because it provides a single number to compare algorithms accounting for both PPV and sensitivity.
Antimalarials included in the medication search were “hydroxychloroquine,” “plaquenil,” “chloroquine,” “quinacrine,” and “aralen.” Oral and intravenous corticosteroids included were “cortisone acetate,” “hydrocortisone,” “Cortef,” “prednisone,” “dexamethasone,” “dexamethasone Intensol,” “decadron,” “prednisolone sodium phosphate,” “Pediapred,” “prednisone Intensol,” “methylprednisolone,” “Medrol,” “Medrol Dosepak,” “prednisolone,” “Orapred,” and “Prelone.” DMARDs included were “azathioprine,” “Imuran,” “methotrexate sodium,” “methotrexate,” “Trexall,” “mycophenolate mofetil,” “CellCept,” “mycophenolic acid,” “Myfortic,” “cyclophosphamide,” “Cytoxan,” “rituximab,” “Rituxan,” “etanercept,” “Enbrel,” “Enbrel Sureclick,” “adalimumab,” “Humira,” “Humira Pen,” “infliximab,” “Remicade,” “abatacept,” and “Orencia.”