A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy

Chen, Huaxian; Kovar, Joy L.; Sissons, Sean E.; Cox, Karen L.; Matter, William F.; Chadwell, Fred; Luan, Peng; Vlahos, Chris J.; Schutz-Geschwender, Amy; Olive, D. Michael

doi:10.1016/j.ab.2004.11.015

Cited by 77 publications

(63 citation statements)

References 17 publications

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“…We have used the ICW or cytoblot to evaluate IRDye 800CW EGF for binding and specifi city prior to actual testing in mice [27] and [55]. In this assay, PC3M-LN4 and 22Rv1 human prostate adenocarcinoma cells were cultured in microtiter plates and treated with serial dilutions of labeled EGF ( Figure 3A) to verify high affi nity binding of EGFR-targeted dye relative to the low binding of free dye alone ( Figure 3B).…”

Section: Testing the Performance And Specifi City Of An Optical Imagimentioning

confidence: 99%

A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models

Kovar

Simpson

Schutz-Geschwender

et al. 2007

Analytical Biochemistry

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141

142

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Section: Testing the Performance And Specifi City Of An Optical Imagimentioning

confidence: 99%

A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models

Kovar

Simpson

Schutz-Geschwender

et al. 2007

Analytical Biochemistry

Self Cite

141

142

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“…The specificity and activity of the IRDye 800CW EGF conjugate were evaluated on PC3M-LN4 and 22Rv1 cells by In-Cell Western, an in vitro cell-based assay. 36 In brief, cells were grown to ϳ90% confluency in a microtiter plate and starved for 2 hours in serum-free (22Rv1) or 1% serum-containing (PC3M-LN4) media. Starvation media were replaced with media containing either IRDye 800CW only (0.002 to 3.3 mol/L), IRDye 800CW EGF (0.01 to 70 nmol/L), unlabeled EGF (0.01 to 2.14 mol/L) plus IRDye 800CW EGF (70 nmol/L), or C225 (0.5 to 200 g/ml) plus IRDye 800CW EGF (70 nmol/L).…”

Section: Characterization Of Egf-irdye 800cw In Vitromentioning

confidence: 99%

Hyaluronidase Expression Induces Prostate Tumor Metastasis in an Orthotopic Mouse Model

Kovar

Johnson

Volcheck

et al. 2006

The American Journal of Pathology

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Molecular mechanisms of prostate cancer progression are frequently studied in mice by orthotopic injection of aggressive cell lines, which yield primary tumors that spontaneously metastasize to lymph nodes. In this report, we characterized the human prostate carcinoma cell line 22Rv1 in an orthotopic system and evaluated the functional relevance of the hyaluronidase Hyal1, a correlate of invasive human prostate cancer, to progression in this model. To provide real-time insights into these processes, we first validated use of an epidermal growth factor-conjugated fluorophore to illuminate orthotopic prostate tumors and their metastases in whole animal imaging. Animals receiving intraprostatic injections were tracked throughout a 6-week period. Tumor sizes were correlated 92% with total fluorescence intensities of 22 prostate tumors. In contrast to the highly tumorigenic and metastatic PC3M-LN4 cells, the 22Rv1 line was orthotopically tumorigenic but not metastatic, despite larger tumor sizes. Lymph node metastasis was successfully imaged in animals with PC3M-LN4 tumors on endpoint dissection. Stable transfection of 22Rv1 cells with Hyal1 did not alter growth kinetics of primary orthotopic tumors, but all animals implanted with Hyal1 transfectants exhibited tumor-positive para-aortic lymph nodes. Hyal1 is implicated as an inducer of prostate cancer metastatic progression.

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“…The binding specifi city of the IRDye 800CW EGF compound was evaluated on A431 and PC3M-LN4 cells by an In-Cell Western assay [23] and [24]. Briefl y, cells were grown to approximately 90% confl uency in a microtiter plate (Nunc, Roberts, WI, USA) and starved for 2 h in serum-free media.…”

Section: Characterization Of Irdye 800cw Egf Binding Specifi City In mentioning

confidence: 99%

Purification method directly influences effectiveness of an epidermal growth factor-coupled targeting agent for noninvasive tumor detection in mice

Kovar

Volcheck

Chen

et al. 2007

Analytical Biochemistry

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This Article is brought to you for free and open access by the Biochemistry, Department of at DigitalCommons@University of Nebraska -Lincoln. It has been accepted for inclusion in Biochemistry --Faculty Publications by an authorized administrator of DigitalCommons@University of NebraskaLincoln.Kovar, Joy L.; Volcheck, William M.; Chen, Jiyan; and Simpson, Melanie A., "Purification method directly influences effectiveness of an epidermal growth factor-coupled targeting agent for noninvasive tumor detection in mice" (2007). Biochemistry --Faculty Publications. 10.

show abstract

A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy

Cited by 77 publications

References 17 publications

A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models

A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models

Hyaluronidase Expression Induces Prostate Tumor Metastasis in an Orthotopic Mouse Model

Purification method directly influences effectiveness of an epidermal growth factor-coupled targeting agent for noninvasive tumor detection in mice

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