Spot Identification and Quality Control in Cell-Based Microarrays

Cell-based microarrays are being increasingly used as a tool for combinatorial and high throughput screening of cellular microenvironments. Analysis of microarrays requires several steps, including microarray imaging, identification of cell spots, quality control, and data exploration. While high content image analysis, cell counting, and cell pattern recognition methods are established, there is a need for new postprocessing and quality control methods for cell-based microarrays used to investigate combinatorial microenvironments. Previously, microarrayed cell spot identification and quality control were performed manually, leading to excessive processing time and potentially resulting in human bias. This work introduces an automated approach to identify cell-based microarray spots and spot quality control. The approach was used to analyze the adhesion of murine cardiac side population cells on combinatorial arrays of extracellular matrix proteins. Microarrays were imaged by automated fluorescence microscopy and cells were identified using open-source image analysis software (CellProfiler). From these images, clusters of cells making up single cell spots were reliably identified by analyzing the distances between cells using a density-based clustering algorithm (OPTICS). Naïve Bayesian classifiers trained on manually scored training sets identified good and poor quality spots using spot size, number of cells per spot, and cell location as quality control criteria. Combined, the approach identified 78% of high quality spots and 87% of poor quality spots. Full factorial analysis of the resulting microarray data revealed that collagen IV exhibited the highest positive effect on cell attachment. This data processing approach allows for fast and unbiased analysis of cell-based microarray data

Pathologic and clinical Alagille syndrome features of 91 patients.

<p>NA: not available.</p><p>*<i>JAG1</i> mutation detected; cases previously reported are in italic font.</p><p>Cases 1–55 met the clinical diagnostic criteria for Alagille syndrome; cases 56–91 were considered as clinically suspected cases.</p><p>Pathologic and clinical Alagille syndrome features of 91 patients.</p

<i>JAG1</i> polymorphisms identified in these cases.

<p>^aidentified in case 67 and maternal;</p><p>^bidentified in case 29 and maternal;</p><p>Novel variant is in bold font.</p><p>dbSNP: single nucleotide polymorphism database; MAF: minor allelic frequency.</p><p><i>JAG1</i> polymorphisms identified in these cases.</p

Summary of <i>JAG1</i> mutations identified in patients.

<p>Novel variants are in bold font; cases previously reported are in italic font. Conserved regions of JAG1 protein include the signal peptide (SP), the delta-serrate-lin12-like region (DSL), epidermal growth factor (EGF)-like repeats, the cysteine-rich (CR) region, and the transmembrane (TM) domain; 5´ of DSL: the region between SP and DSL domain; 5´ of TM: the region between CR and TM.</p><p>het: heterozygous; MPLA: multiplex-ligation-dependent probe amplification; ND: not done.</p><p>Summary of <i>JAG1</i> mutations identified in patients.</p

Aggregate survival tests <i>in vitro</i>.

<p><b>A)</b> Subsets of microwell arrays with 2D monolayer of cell culture (2D) and aggregates of three sizes (S, M, and L). Hydrogen peroxide and anoxia/reoxygenation treatments were employed to induce cell death. EthD (red) and DAPI (blue) staining were performed for the determination of cell death. <b>B)</b> Quantification of dead CSP cells in 2D single layer culture and aggregates with variable diameters subjected to 200 µM-hydrogen peroxide treatment using EthD/DAPI fluorescent intensity ratio. Data were normalized to the vehicle groups of 2D monolayer culture and aggregates in three sizes. <b>C)</b> Quantification of dead CSP cells in 2D single layer culture and aggregates with variable diameters subjected to anoxia/reoxygenation using EthD/DAPI fluorescent intensity ratio. Data were normalized to the vehicle groups of 2D monolayer culture and aggregates in three sizes.</p

CSP cell survival <i>in vivo</i> following cardiac injury.

<p><b>A)</b> Protocol to measure the <i>in vivo</i> survival of CSP aggregates and suspensions. <b>B)</b> Representative serial bioluminescence images (BLI) of mice injected with CSP cell aggregates and CSP single cell suspensions. <b>C)</b> Percentage of CSP cell survival measured with BLI.</p

Aggregate integrity and survival in fluidic manipulations.

<p><b>A)</b> Aggregates formed in microwells can be easily flushed out from the microwell and centrifuged while remaining intact. <b>B)</b> Aggregate can be easily passed through a 30G needle without loosing integrity. <b>C)</b> A representative DAPI/EthD fluorescent image of aggregates before injection. <b>D)</b> A representative DAPI/EthD fluorescent image of aggregates after injection. <b>E)</b> Quantification of dead CSP cells in aggregates passing a 30G needle using EthD/DAPI fluorescent intensity ratio. (All bars represent 100 µm).</p

Cell patterning and aggregate formation inside microwells.

<p><b>A)</b> Cell patterning. Cells were localized inside the microwells. <b>B)</b> After cell seeding, the cells in the microwell array were cultured in a petri dish and aggregates formed within 24 h. <b>C)</b> Once the aggregate formation is complete inside the microwells, they can be stained. <b>D)</b> Aggregates can be imaged inside microwells. <b>E)</b> Aggregates can be easily released from the microwells by gentle flushing with media for other applications.</p

Activation of NFκB mediates TWEAK-induced downregulation of PGC1α.

<p>(A) Immunoblots of phospho-p65, phospho-IκBα, total-IκBα and GAPDH in isolated cardiomyocytes incubated with 100 ng/ml IgG or rTWEAK at designated time points. (B) Inhibition of NFκB activation with SC-514 (25µM) abolished TWEAK-mediated downregulation of PGC1α expression. * p<0.05 vs. IgG and # p<0.05 vs. rTWEAK in the absence of SC-514.</p

rTWEAK delivery results in cardiomyopathy and altered metabolic gene expression.

<p>(A) Fractional shortening (FS%) and (B) Left ventricular diastolic chamber dimension (LVID), as determined by transthoracic echocardiography in mice one week after intravenous delivery of IgG or rTWEAK. (C) Representative M-Mode echocardiographic images before and following IgG or rTWEAK injection in mice. Real time PCR analysis of heart samples for expression of (D) PGClα and (E) OXPHOS genes, normalized to β-actin and presented relative to the IgG group. (F) Mitochondria membrane potential in IgG and rTWEAK treated cardiomyocytes. The membrane potential was measured by TMRE staining. The fluorescent intensities of TMRE were normalized to the total number of cardiomyocytes in the respective fields. * p<0.05 vs. IgG, N = 3 for each group.</p