Real-Time Chemotaxis of Bio-Gel Elicited Macrophages to Murine CCL5
(A)Representative trace from the RTCA software of 4×105 Bio-Gel elicited macrophages from C57BL/6J in the top chamber with murine CCL5 (5nM) (red) or no chemokine (green) in the bottom chamber. A control of no macrophages and no chemokine was also run (blue). By 70mins max CI had been reached, monitoring the plate was stopped, removed and membranes were cut and fixed with 2.5% gluteraldehyde and processed for scanning electron microscopy (SEM). Representative images are shown of macrophages adhered to the underside of fi lters in response to 5 nM murine CCL5 (B,C)no chemokine (D,E)or control where no cells or chemokine were added(F). The number of cells adhered to individual gold discs on the underside of the membrane (seen as light grey regions in the SEM images) were quantitated (n=1 experiment with 5-6 technical replicates of each condition analysed) (G) (Data and figures adapted from Iqbal AJ, et al., 2013).
Assessment of Ovarian Cancer Spheroid Invasion of Mesothelial Cells
(A) Spheroid generation and model of experimental set up. Schematic showing the RTCA 2 chambered CIM plate well set up, in which pre-formed ovarian cancer spheroids are plated on top of a monolayer of an LP9 mesothelial layer/matrix barrier in the upper chamber and media ±FBS as a chemoattractant is added to the lower chamber. Electrodes underneath the interface of the 2 chambers measure increasing electrical impedance as more cells invade through the barriers to the lower chamber. (B - C) Representative results from an RTCA invasion assay conducted with and without FBS in the bottom chamber of a CIM plate well. KGN cell invasion is compared to LP9 mesothelial cells. Results are shown as mean ± SD Cell Index from triplicate wells at the 24 hr timepoint (B)and over an entire two day assay period(C). (Data and figures adapted from Bilandzic M & Stenvers KL, 2014).
Select Publications For Cell Invasion And Migration
1.Assessment of ovarian cancer spheroid attachment and invasion of mesothelial cells in real time.
Bilandzic M, Stenvers KL.
J Vis Exp. 2014 May 20;(87).
2.A real time chemotaxis assay unveils unique migratory profiles amongst different primary murine macrophages.
Iqbal AJ, Regan-Komito D, Christou I, White GE, McNeill E, Kenyon A, Taylor L, Kapellos TS, Fisher EA, Channon KM, Greaves DR.
PLoS One. 2013;8(3):e58744.
3.c-Myb regulates matrix metalloproteinases 1/9, and cathepsin D: implications for matrix- dependent breast cancer cell invasion and metastasis.
Knopfová L, Beneš P, Pekarcíková L, Hermanová M, Masařík M, Pernicová Z, Souček K,Smarda J.
Molecular Cancer. 2012 Mar 23;11:15.
4.MicroRNA-200c Represses Migration and Invasion of Breast Cancer Cells by Targeting Actin- Regulatory Proteins FHOD1 and PPM1Ferences.
Jurmeister S, Baumann M, Balwierz A, Keklikoglou I, Ward A, Uhlmann S, Zhang JD, Wiemann S, Sahin Ö.
Molecular and Cellular Biology. 2012 Feb;32(3):633-51.
5.Comparative Analysis of Dynamic Cell Viability, Migration and Invasion Assessments by Novel Real-Time Technology and Classic Endpoint Assays.
Limame R, Wouters A, Pauwels B, Fransen E, Peeters M, Lardon F, De Wever O, Pauwels P.
PLoS One. 2012;7(10):e46536.
• Quantitative monitoring of cell migration or invasion in real time for up to hundreds hours.
• Label-free assay requires no fixation, staining or any other sample processing,dramatically reducing hands-on time.
• Easy quantification of the kinetics of migration or invasion.
• Rapid optimization of cell density and extracellular matrix density conditions.
• Non-invasive nature allows for quantitation of migrated/invaded cells by standard assays upon completion, typically revealing excellent correlation between Cell Index and migrated/invaded cell numbers.