FLIPR: A New Instrument for Accurate, High Throughput Optical Screening

Schroeder, Kirk S.; Neagle, Brad

doi:10.1177/108705719600100205

Cited by 190 publications

(115 citation statements)

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“…Therefore, we used the Fluorometric Imaging Plate Reader (FLIPR) system, a new high-throughput screening tool for cell-based fluorescent assays (28). The unique aspect of the FLIPR is that it simultaneously reads all the wells of a standard 96-well microplate, with kinetic updates in the subsecond range.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of SDF‐1/CXCR4‐induced Ca²⁺ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

et al. 2002

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BackgroundThe chemokine receptors CXCR4 and CCR5 are the main coreceptors for human immunodeficiency virus (HIV) 1 to enter its target cells. The antiviral activity of their natural ligands (stromal cell‐derived factor 1 [SDF‐1], regulated on activation normal T‐cell expressed and secreted, (RANTES) and macrophage inflammatory proteins 1α and 1β, MIP‐1α and MIP‐1β) and the finding that individuals deficient in CCR5 are relatively resistant to HIV infection led to the concept that chemokine receptor antagonists can play an important role in anti‐HIV therapy. AMD3100, the prototype compound of the bicyclams, is one of the most potent and selective CXCR4 antagonists described to date. The search for new chemokine receptor antagonists involves the evaluation of compounds for their ability to block the specific chemokine‐induced transient intracellular Ca2+ flux. We evaluated two cell‐based‐fluorescent methods with the use of the Fluorometric Imaging Plate Reader (FLIPR) system and a flow cytometric assay to measure the SDF‐1–induced intracellular Ca2+ mobilization via CXCR4. Both assay systems were compared for their sensitivity, advantages, and system‐dependent limitations.MethodsCXCR4+ lymphocytic and monocytic cell lines commonly used in laboratories studying acquired immunodeficiency syndrome and freshly isolated peripheral blood mononuclear cells (PBMCs) were loaded with the Ca2+ indicator Fluo‐3. After washing, the cells were preincubated with the CXCR4 antagonist AMD3100. Then the increase in intracellular Ca2+ concentration after stimulation with SDF‐1 was examined by the FLIPR and the flow cytometer, which monitored the change in green fluorescence intensity of Ca2+‐bound Fluo‐3. Surface CXCR4 expression was also determined flow cytometrically.ResultsIn all five CXCR4+ cell lines, SDF‐1 elicited a transient increase in intracellular Ca2+ concentration. For each cell line, the magnitude of response was related to the level of CXCR4 expression: the cells with the highest CXCR4 level showed the strongest Ca2+ response. AMD3100 effected a dose‐dependent inhibition of the SDF‐1–induced Ca2+ flux in all cell lines examined. The FLIPR was more sensitive than the flow cytometer in detecting minor Ca2+ responses. In freshly isolated PBMCs, the Ca2+ response was due solely to the stimulation of monocytes and granulocytes, whereas the lymphocyte population, although expressing CXCR4, did not respond at all. This phenomenon could be observed with the flow cytometer and not with the FLIPR.ConclusionsThe FLIPR system is a most adequate to monitor intracellular Ca2+ mobilization and to evaluate chemokine receptor antagonists. However, flow cytometry and its multiparameter analysis allow additional characterization of the cells involved in the chemokine receptor‐mediated signal transduction. Cytometry Part A 51A:35–45, 2003. © 2002 Wiley‐Liss, Inc.

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Section: Discussionmentioning

confidence: 99%

Evaluation of SDF‐1/CXCR4‐induced Ca²⁺ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

et al. 2002

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“…The final aspiration from the Denley cell washer was adjusted to leave 150 l of residual buffer (same settings as above except soak ϭ 0 and volume ϭ 0). The microtiter plate was placed in a fluorescence-imaging plate reader (FLIPR; Molecular Devices, Sunnyvale, CA) that determined cell-associated fluorescence simultaneously in all wells of a 96-well microtiter plate maintained at 37°C (24). An additional plate was prepared that contained triplicate wells of stimulus dissolved in Ca 2ϩ flux buffer (MCP-1 dilutions, ionomycin and buffer alone).…”

Section: Fluorometric Imaging Plate Reader Ca 2ϩ Flux Assaymentioning

confidence: 99%

Functional Differences Between Monocyte Chemotactic Protein-1 Receptor A and Monocyte Chemotactic Protein-1 Receptor B Expressed in a Jurkat T Cell

Sanders

Crean

Boxer

et al. 2000

The Journal of Immunology

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The monocyte chemotactic protein-1 (MCP-1) receptor (MCP-1R) is expressed on monocytes, a subpopulation of memory T lymphocytes, and basophils. Two alternatively spliced forms of MCP-1R, CCR2A and CCR2B, exist and differ only in their carboxyl-terminal tails. To determine whether CCR2A and CCR2B receptors function similarly, Jurkat T cells were stably transfected with plasmids encoding the human CCR2A or CCR2B gene. Nanomolar concentrations of MCP-1 induced chemotaxis in the CCR2B transfectants that express high, intermediate, and low levels of MCP-1R. Peak chemotactic activity was shifted to the right as receptor number decreased. Five-fold more MCP-1 was required to initiate chemotaxis of the CCR2A low transfectant, but the peak of chemotaxis was similar for the CCR2A and CCR2B transfectants expressing similar numbers of receptors. MCP-1-induced chemotaxis was sensitive to pertussis toxin, implying that both CCR2A and CCR2B are Giα protein coupled. MCP-1 induced a transient Ca2+ flux in the CCR2B transfectant that was partially sensitive to pertussis toxin. In contrast, MCP-1 did not induce Ca2+ flux in the CCR2A transfectant. Since MCP-1 can stimulate chemotaxis of the CCR2A transfectant without inducing Ca2+ mobilization, Ca2+ flux may not be required for MCP-1-induced chemotaxis in the Jurkat transfectants. These results indicate that functional differences exist between the CCR2A and CCR2B transfectants that can be attributed solely to differences in the carboxyl-terminal tail.

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“…Measurement of Intracellular pH Using BCECF-We used a FLIPR™ fluorometric imaging plate reader system (27) to measure intracellular pH in CHO-K1 cells. Cells were seeded (ϳ 50,000 cells/ well) in 96-well clear bottom black microplates (Corning Costar Corp., Cambridge, MA) and left overnight in CO 2 incubator at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Hypertonicity Activates Na+/H+ Exchange through Janus Kinase 2 and Calmodulin

Garnovskaya

Mukhin

Vlasova

et al. 2003

Journal of Biological Chemistry

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The type 1 sodium-hydrogen exchanger (NHE-1) is a ubiquitous electroneutral membrane transporter that is activated by hypertonicity in many cells. NHE-1 may be an important pathway for Na ؉ entry during volume restoration, yet the molecular mechanisms underlying the osmotic regulation of NHE-1 are poorly understood. In the present study we conducted a screen for important signaling molecules that could be involved in hypertonicity-induced activation of NHE-1 in CHO-K1 cells. Hypertonicity rapidly activated NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry and by measurements of intracellular pH on a FLIPR TM (fluorometric imaging plate reader). Inhibitors of Ca 2؉ /calmodulin (CaM) and Janus kinase 2 (Jak2) attenuated this activation, whereas neither calcium chelation nor inhibitors of protein kinase C, the Ras-ERK1/2 pathway, Src kinase, and Ca 2؉ /calmodulindependent enzymes had significant effects. Hypertonicity also resulted in the rapid tyrosine phosphorylation of Jak2 and STAT3 (the major substrate of Jak2) and CaM. Phosphorylation of Jak2 and CaM were blocked by AG490, an inhibitor of Jak2. Immunoprecipitation studies showed that hypertonicity stimulates the assembly of a signaling complex that includes CaM, Jak2, and NHE-1. Formation of the complex could be blocked by AG490. Thus, we propose that hypertonicity induces activation of NHE-1 in CHO-K1 cells in large part through the following pathway: hypertonicity 3 Jak2 phosphorylation and activation 3 tyrosine phosphorylation of CaM 3 association of CaM with NHE-1 3 NHE-1 activation.The ubiquitous isoform of the Na ϩ /H ϩ exchanger (NHE-1) 1 is essential for the regulation of cellular volume and intracellular pH. NHE-1 is nearly quiescent in resting cells but is activated by a variety of hormones and growth factors (1, 2). NHE-1 is also rapidly activated by hypertonic stress in many cells (3), and this may be an important pathway for Na ϩ entry during volume restoration. Despite the potential importance of this process, the molecular mechanisms underlying the regulation of NHE-1 by hypertonicity have not been fully elucidated. The rapid activation of NHE-1 is often associated with an increase in its phosphorylation (3). Kinases that have been shown to directly phosphorylate NHE-1 include p90 S6 kinase (4) and the Nck-interacting kinase (5). However, deletion of the major phosphorylation sites contained within residues 636 -815 of NHE-1 only reduces its response to growth factors by about 50% (6), suggesting that mechanisms of regulation other than direct phosphorylation of NHE-1 are also important.Hypertonicity-induced shrinkage of mammalian cells is a powerful stimulant for many protein kinases that could play important direct or indirect roles in activating NHE-1. These include mitogen-activated protein kinases such as extracellular signal-regulated protein kinase (ERK), stress-activated protein kinases (c-Jun N-terminal kinases) (7-10), Src family tyrosine kinases p59 fgr and p56/59 hck (11), protein kinase C (12-14), Jan...

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FLIPR: A New Instrument for Accurate, High Throughput Optical Screening

Cited by 190 publications

References 2 publications

Evaluation of SDF‐1/CXCR4‐induced Ca²⁺ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

Evaluation of SDF‐1/CXCR4‐induced Ca²⁺ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

Functional Differences Between Monocyte Chemotactic Protein-1 Receptor A and Monocyte Chemotactic Protein-1 Receptor B Expressed in a Jurkat T Cell

Hypertonicity Activates Na+/H+ Exchange through Janus Kinase 2 and Calmodulin

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FLIPR: A New Instrument for Accurate, High Throughput Optical Screening

Cited by 190 publications

References 2 publications

Evaluation of SDF‐1/CXCR4‐induced Ca2+ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

Evaluation of SDF‐1/CXCR4‐induced Ca2+ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

Functional Differences Between Monocyte Chemotactic Protein-1 Receptor A and Monocyte Chemotactic Protein-1 Receptor B Expressed in a Jurkat T Cell

Hypertonicity Activates Na+/H+ Exchange through Janus Kinase 2 and Calmodulin

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Resources

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Evaluation of SDF‐1/CXCR4‐induced Ca²⁺ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

Evaluation of SDF‐1/CXCR4‐induced Ca²⁺ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry