reSOLUTION_Research_09_Neuroscience - Leica Microsystems

HigH content screening
CYTOO’s HCA Platform and Leica HCS A
Normalize cells
dr. constantin nelep and dr. Joanne young, cytoo cell architects
the combination of cytoo’s Hca Platform with the leica Hcs a solution provides a highly reproducible high content analysis solution.
conventional cell culture conditions on uniform adhesive supports lead to high morphological variability and uncontrolled cell migration.
this situation is in sharp contrast to what is found in tissue, where cells respond to external spatial information (both from the extracellular
matrix and neighboring cells) and adopt a reproducible polarized architecture, necessary for proper tissue function.
fig. 1: cytoo micropatterned supports
come in a range of: (a) cytoochips
(up to 20,000 micropatterns per coverslip
with special cell localization grid) and
(b) cytooplates (up to 5000 micropattern
per well) dedicated respectively for
basic research and for High-throughput
microscopy/Hcs applications. standard
micropattern shapes include crossbow,
H, y, l and full disks.
fig. 2: example of micropatterned Hela
single cells (seeded on 1100 µm 2 fibronectin
crossbow-shape patterns providing
cell polarization). cytoochip imaged
with leica tsc sPe / las af matrix.
objective 63x. actin: Phalloidin-fitc;
nuclei: Hoechst. only 20 – 50 cells are
needed to obtain statistically relevant
data.
24 resolutioN
a
b
Highly reproducible results
advanced cell culture supports developed by cytoo
cell architects (fig. 1) use proprietary concave
adhesive micropatterns that control the cell adhesion
microenvironment. as cells show a highly reproducible
response to the geometry of the adhesive micropattern,
this leads to the normalization of cell shape,
internal architecture 1 and orientation of division 2
(fig. 2) thereby overcoming a fundamental obstacle
that plagues all current High content studies.
exquisite control over assay design
as micropattern features (shape, size, molecular
composition) can be modulated and refined in myriad
ways, live and endpoint analyses, both existing and
novel ones, can be engineered for a wide range of
cellular activities, processes and phenotypes, thereby
enabling much greater High content data depth.
the reference cell
this dramatic decrease in cell variability enables
highly reproducible quantification of the spatial distribution
of cell compartments and individual proteins 1,3 .
averaging the distribution observed across only a
limited number of cells (20 – 50) leads to a reference
cell, a highly robust representation of protein and
organelle organization. the reference cell can be
used to assess the significance of any perturbation
introduced in and across large scale screening experiments
and reveals any subtle changes in cellular
phenotype 2 . moreover, the regular array of adhesive
micropatterns significantly facilitates automated
image capture microscopy both with fixed and live
cells and resolves the cell segmentation headaches
encountered with image analysis of cells grown on
conventional cell culture supports. integration of
micropattern specific image processing with interactive
microscope control can also provide powerful
and flexible experimental designs such as on-the-fly
image analysis for capturing rare events.
References
1. thery, m. et al. anisotropy of cell adhesive microenvironment governs
cell internal organization and orientation of polarity. Proc natl
acad sci u s a 103, 19771-19776 (2006).
2. thery, m. et al. the extracellular matrix guides the orientation of
the cell division axis. nat cell biol 7, 947-953 (2005).
3. schauer, K. et al. Probabilistic density maps to study global endomembrane
organization. nat methods (2010).
Contact
dr. constantin nelep
cytoo cell architects
cnelep@cytoo.com
www.cytoo.com