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Total internal reflection fluorescence quantification of receptor pharmacology.

Fang Y - Biosensors (Basel) (2015)

Bottom Line: Total internal reflection fluorescence (TIRF) microscopy has been widely used as a single molecule imaging technique to study various fundamental aspects of cell biology, owing to its ability to selectively excite a very thin fluorescent volume immediately above the substrate on which the cells are grown.Inspired by the recent demonstration of label-free evanescent wave biosensors for cell phenotypic profiling and drug screening with high throughput, we had hypothesized and demonstrated that TIRF imaging is also amenable to receptor pharmacology profiling.This paper reviews key considerations and recent applications of TIRF imaging for pharmacology profiling.

View Article: PubMed Central - PubMed

Affiliation: Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, NY 14831, USA. fangy2@corning.com.

ABSTRACT
Total internal reflection fluorescence (TIRF) microscopy has been widely used as a single molecule imaging technique to study various fundamental aspects of cell biology, owing to its ability to selectively excite a very thin fluorescent volume immediately above the substrate on which the cells are grown. However, TIRF microscopy has found little use in high content screening due to its complexity in instrumental setup and experimental procedures. Inspired by the recent demonstration of label-free evanescent wave biosensors for cell phenotypic profiling and drug screening with high throughput, we had hypothesized and demonstrated that TIRF imaging is also amenable to receptor pharmacology profiling. This paper reviews key considerations and recent applications of TIRF imaging for pharmacology profiling.

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Label-free cell phenotypic assays for decoding receptor composition and signaling. (a) A hypothetical kinetic response of living cells on the sensor surface; (b) The real-time DMR dose responses of pinacidil in HepG2C3A cells; (c–f) The DMR of 40 µM pinacidil in the mock- transfected cells, in comparison with those in cells treated with three different SUR1 RNAi (c); SUR2 RNAi (d); Kir6.1 RNAi (e); or Kir6.2 RNAi (f); (g) The DMR of 32 µM pinacidil as a function of different KATP blockers; (h) The sensitivity of 32 µM pinacidil DMR to kinase inhibition, plotted as its amplitudes at 50 min post stimulation as a function of compound; (i) The real-time DMR of 40 µM pinacidil with mock transfection (mock) or JAK siRNA; (j) The real-time DMR of 40 µM pinacidil with mock transfection (mock) or ROCK siRNA. For (c–f) and (i–j), RNAi transfection was done 48hrs prior to the pinacidil stimulation. For (g,h), the cells were first treated with each blocker for 1hr, followed by stimulation with 32 µM pinacidil. For (i) and (j), the buffer DMR was included as a negative control. Data represents mean ± s.d. (n = 6 for a–f, n = 3 for g, n = 4 for h, n = 6 for i and j). This figure is reproduced from Ref. [14] through the Creative Commons Attribution License.
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biosensors-05-00223-f004: Label-free cell phenotypic assays for decoding receptor composition and signaling. (a) A hypothetical kinetic response of living cells on the sensor surface; (b) The real-time DMR dose responses of pinacidil in HepG2C3A cells; (c–f) The DMR of 40 µM pinacidil in the mock- transfected cells, in comparison with those in cells treated with three different SUR1 RNAi (c); SUR2 RNAi (d); Kir6.1 RNAi (e); or Kir6.2 RNAi (f); (g) The DMR of 32 µM pinacidil as a function of different KATP blockers; (h) The sensitivity of 32 µM pinacidil DMR to kinase inhibition, plotted as its amplitudes at 50 min post stimulation as a function of compound; (i) The real-time DMR of 40 µM pinacidil with mock transfection (mock) or JAK siRNA; (j) The real-time DMR of 40 µM pinacidil with mock transfection (mock) or ROCK siRNA. For (c–f) and (i–j), RNAi transfection was done 48hrs prior to the pinacidil stimulation. For (g,h), the cells were first treated with each blocker for 1hr, followed by stimulation with 32 µM pinacidil. For (i) and (j), the buffer DMR was included as a negative control. Data represents mean ± s.d. (n = 6 for a–f, n = 3 for g, n = 4 for h, n = 6 for i and j). This figure is reproduced from Ref. [14] through the Creative Commons Attribution License.

Mentions: In recent years, label-free optical biosensors such as RWG and SPR have been used to study many fundamental aspects of cell biology including cell adhesion, cycle, proliferation, receptor signaling, death, cell barrier functions, cell-to-cell communication, migration, invasion, differentiation, and viral infection (reviewed in [68,69,70,71,72,73,74,75]). This is due to the ability of these biosensors to non-invasively monitor the entire life cycle of living cells on the sensor surface (Figure 4a). These biosensors offer great flexibility in assay formats, so a wide range of cell phenotypes can be studied [19,71,73]. In particular, the RWG biosensor has been found to be a powerful high-throughput technique for receptor biology studies [10,11,12,13,14,20,21] and drug profiling and screening [15,16,17,18,19,22,75,76].


Total internal reflection fluorescence quantification of receptor pharmacology.

Fang Y - Biosensors (Basel) (2015)

Label-free cell phenotypic assays for decoding receptor composition and signaling. (a) A hypothetical kinetic response of living cells on the sensor surface; (b) The real-time DMR dose responses of pinacidil in HepG2C3A cells; (c–f) The DMR of 40 µM pinacidil in the mock- transfected cells, in comparison with those in cells treated with three different SUR1 RNAi (c); SUR2 RNAi (d); Kir6.1 RNAi (e); or Kir6.2 RNAi (f); (g) The DMR of 32 µM pinacidil as a function of different KATP blockers; (h) The sensitivity of 32 µM pinacidil DMR to kinase inhibition, plotted as its amplitudes at 50 min post stimulation as a function of compound; (i) The real-time DMR of 40 µM pinacidil with mock transfection (mock) or JAK siRNA; (j) The real-time DMR of 40 µM pinacidil with mock transfection (mock) or ROCK siRNA. For (c–f) and (i–j), RNAi transfection was done 48hrs prior to the pinacidil stimulation. For (g,h), the cells were first treated with each blocker for 1hr, followed by stimulation with 32 µM pinacidil. For (i) and (j), the buffer DMR was included as a negative control. Data represents mean ± s.d. (n = 6 for a–f, n = 3 for g, n = 4 for h, n = 6 for i and j). This figure is reproduced from Ref. [14] through the Creative Commons Attribution License.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4493547&req=5

biosensors-05-00223-f004: Label-free cell phenotypic assays for decoding receptor composition and signaling. (a) A hypothetical kinetic response of living cells on the sensor surface; (b) The real-time DMR dose responses of pinacidil in HepG2C3A cells; (c–f) The DMR of 40 µM pinacidil in the mock- transfected cells, in comparison with those in cells treated with three different SUR1 RNAi (c); SUR2 RNAi (d); Kir6.1 RNAi (e); or Kir6.2 RNAi (f); (g) The DMR of 32 µM pinacidil as a function of different KATP blockers; (h) The sensitivity of 32 µM pinacidil DMR to kinase inhibition, plotted as its amplitudes at 50 min post stimulation as a function of compound; (i) The real-time DMR of 40 µM pinacidil with mock transfection (mock) or JAK siRNA; (j) The real-time DMR of 40 µM pinacidil with mock transfection (mock) or ROCK siRNA. For (c–f) and (i–j), RNAi transfection was done 48hrs prior to the pinacidil stimulation. For (g,h), the cells were first treated with each blocker for 1hr, followed by stimulation with 32 µM pinacidil. For (i) and (j), the buffer DMR was included as a negative control. Data represents mean ± s.d. (n = 6 for a–f, n = 3 for g, n = 4 for h, n = 6 for i and j). This figure is reproduced from Ref. [14] through the Creative Commons Attribution License.
Mentions: In recent years, label-free optical biosensors such as RWG and SPR have been used to study many fundamental aspects of cell biology including cell adhesion, cycle, proliferation, receptor signaling, death, cell barrier functions, cell-to-cell communication, migration, invasion, differentiation, and viral infection (reviewed in [68,69,70,71,72,73,74,75]). This is due to the ability of these biosensors to non-invasively monitor the entire life cycle of living cells on the sensor surface (Figure 4a). These biosensors offer great flexibility in assay formats, so a wide range of cell phenotypes can be studied [19,71,73]. In particular, the RWG biosensor has been found to be a powerful high-throughput technique for receptor biology studies [10,11,12,13,14,20,21] and drug profiling and screening [15,16,17,18,19,22,75,76].

Bottom Line: Total internal reflection fluorescence (TIRF) microscopy has been widely used as a single molecule imaging technique to study various fundamental aspects of cell biology, owing to its ability to selectively excite a very thin fluorescent volume immediately above the substrate on which the cells are grown.Inspired by the recent demonstration of label-free evanescent wave biosensors for cell phenotypic profiling and drug screening with high throughput, we had hypothesized and demonstrated that TIRF imaging is also amenable to receptor pharmacology profiling.This paper reviews key considerations and recent applications of TIRF imaging for pharmacology profiling.

View Article: PubMed Central - PubMed

Affiliation: Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, NY 14831, USA. fangy2@corning.com.

ABSTRACT
Total internal reflection fluorescence (TIRF) microscopy has been widely used as a single molecule imaging technique to study various fundamental aspects of cell biology, owing to its ability to selectively excite a very thin fluorescent volume immediately above the substrate on which the cells are grown. However, TIRF microscopy has found little use in high content screening due to its complexity in instrumental setup and experimental procedures. Inspired by the recent demonstration of label-free evanescent wave biosensors for cell phenotypic profiling and drug screening with high throughput, we had hypothesized and demonstrated that TIRF imaging is also amenable to receptor pharmacology profiling. This paper reviews key considerations and recent applications of TIRF imaging for pharmacology profiling.

Show MeSH