4. Cytokinesis following mitosisMembrane Ruffling

5. The basic principle of ECIS was first reported by Giaever and Keese, then at the General Electric Corporate Research and Development Center. Giaever, I. And Keese, C.R. PNAS 81, 3761-3764 (1984). ECIS  Electric Cell-substrate Impedance Sensing

6. 250 µm WE CE WE: Working Electrode CE: Counter Electrode The ECIS Electrodes

7. 250  m Array Holder in Incubator Space ECIS 8 well Array

8. ECIS  Electric Cell-substrate Impedance Sensing A cell morphology biosensor <1  A, 4000 Hz The measurement is non-invasive AC Current source PC ECIS electrode Counter electrode Culture medium (electrolyte) Phase sensitive impedance measurement PC R C

9. Cell Inoculation (10 5 cells per cm 2 ) BSC-1cells NRK cells No cells

10. A published model fits the experimental data The measured impedance can be broken down into three parameters 1) Rb, the barrier function of the cell layer 2) Alpha, a term associated with the constricted current flow beneath the cell 3) Cm, the membrane capacitance [Giaever, I. and Keese, C.R., PNAS 81, 3761 (1991)]

11. Detection of single cell activity

12. What is measured using ECIS? Cell morphology changes including: 1) Barrier function of confluent layers 2) Relative size of cells and spaces beneath cells 3) Membrane capacitance All measurements are made in normal culture medium The measurement is non- invasive Limitations Cells must anchor and spread upon substratum A limited population of cells is measured at one time (1 to 1,000 cells)

13. DNA RNA Viral Infection Glucose Oxygen COOH OOCCH 3 Drugs Ligand Binding Physical Changes Shear, Electric Fields Changes in Cell Morphology Metabolism Cytoskeleton Electric Cell-Substrate Impedance Sensing

14. Measurement of Metastatic Potential using ECIS™ BioTechniques, October 2002 Keese, Bhawe, Wegener and Giaever

15. The basis of the metastatic assay

16. The Dunning prostatic adenocarcinoma series was developed at Johns Hopkins and consists of several cell sublines. These all have their origin in a single line isolated from a prostatic tumor. After extensive passaging and mutagenesis, several distinct sublines were isolated having different in vivo metastatic abilities. Six of these lines were used in our studies.

17. To carry out the metastatic assay, first a layer of endothelial cells is established Confluence verified

18. Challenge of HUVEC cell layers with weakly (G) and highly metastatic (AT3) cell lines Challenge highly metastatic

19. Confluent HUVEC layer No cells MLL Challenge 10 5 cells/cm 2

20. Prostatic cell challenge

21. Signal Transduction

22.  [Ca 2+ ] Alterations in the cytoskeleton G Protein Coupled Receptor

23. CHO cells engineered to over- express the muscarinic receptor exposed to the agonist carbachol EC 50 = ~1  M

24. The effect of carbachol is blocked by the antagonist pirenzipine (PZP )

26. Treatment of CHO-M1T cells with carbachol Data analysis using the ECIS model  morphological information

27. Similar results are obtained with the beta adrenergic receptor

28. The Dynamics of Cell Spreading

29. WI-38 VA/13 cells Cell inoculation 10 5 cells/cm 2 Electrodes were pre- coated with different layers of adsorbed protein before cell inoculation Adsorbed proteins alter cell spreading dynamics

30. MDCK II cells inoculated on electrodes pre-coated with various proteins FN fibronectin LAM laminin VN vitronectin BSA bovine serum albumin BSA FN Inoculation Confluent Cell-free Capacitance at high freq. measures the open (cell- free) electrode area

31. Adsorb BSA re-inoculate with MDCK cells after 24 hours remove cell MDCK cells BSA is adsorbed to the electrodes and they are inoculated with MDCK cells

32. Adsorb BSA re-inoculate with MDCK cells after 24 hours remove cell MDCK cells Laminin-like response

33. MDCK cells inoculated on fibronectin-coated electrodes with different concentrations of synthetic tetrapeptide RGDS present

34. MDCK cells inoculated on laminin-coated electrodes with different concentrations of synthetic tetrapeptide RGDS present

35. Elevated Field Applications 1 Electroporation 2 Wound healing assay

36. Elevated Field Applications 1 Electroporation 2 Wound healing assay

37. NORMAL MODE 1 MICROAMP, 10 MILLIVOLTS ELEVATED FIELD 1 MILLIAMP, A FEW VOLTS pore formation Elevated current applied ~200msec

38. 500 msec200 msec100 msec50 msec Variation of the pulse duration: Lucifer yellow uptake Pulse:40 kHz 4.0 V MDCK Type II cells Variation of the pulse duration: Lucifer yellow uptake Pulse:40 kHz 4.0 V MDCK Type II cells

39. Uptake of dyes with different molecular weight Lucifer Yellow M = 0.5 kDa TRITC-dextran M = 76 kDa Pulse:40 kHz, 4.0 V, 200 msec FITC-dextran M = 250 kDa Albany Medical College (F. Minnear) has demonstrated introduction of DNA constructs using the method and obtained expression of GFP

40. bleomycin only bleomycin with electroporation High field pulse for 100 msec Electroporated control Electroporation of bleomycin into HUVEC monolayers

41. Wound Healing (migration) Assay

42. Traditional Wound Healing Assay Problems of reproducibility and quantification Cell migration

43. 500 msec200 msec100 msec50 msec Variation of the pulse duration: Lucifer yellow uptake Pulse:40 kHz 4.0 V MDCK Type II cells Cell death

44. NORMAL MODE 1 MICROAMP, 10 MILLIVOLTS ELEVATED FIELD 1 MILLIAMP, A FEW VOLTS Severe pore formation localized heating Elevated current applied 15 seconds CELL WOUNDING

45. NRK Cells Prior to Wounding

46. NRK Cells Immediately after Wounding

47. NRK Cells During Healing

48. NRK Cells After Healing

49. Confluence Open electrode RPI

50. BSC-1 cells NRK cells wounding

51. Phase Contrast Microscopy of MDCK Cell Wounding CONTROL WOUNDED20 HOURS LATER Are the cells killed, or are they simply damaged and recovering?

52. Calcein-AM and Ethidium Staining Control3 V, 10 sec

53. BSC-1 cells wounded on different size electrodes Standard 250 micron diameter electrode wound

54. BSC-1 cells wounded on different size electrodes 100 microns wound

55. BSC-1 cells wounded on different size electrodes 50 microns wound

56. BSC-1 cells wounded on different size electrodes Lag period migration = ~17 microns/hr

57. Phase Contrast Microscopy of MDCK Cell Wounding CONTROL WOUNDED20 HOURS LATER

59. Initial wound Re-wound The approach is highly reproducible

60. New directions Flow cell for endothelial cell studies 96 well Format for HTS

61. ECIS  9600

62. ECIS  Flow System

63. Acknowledgements: Ivar Giaever President of Applied BioPhysics and Institute Professor at Rensselaer Joachim Wegener Sarah Walker, Kaumudi Bhawe, Steve Tet, Will Wu, Lali Reddy, Paramita Ghosh, Guo Chen, Narayan Karra Funding from: NIH SBIR Program NCRR NCI NIEHS National Foundation for Cancer Research

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