|
 Assay
description:
The cell cycle
is commonly divided into “phases” – interphase and mitosis.
Interphase is further divided into three sub-phases, G1, S, and
G2. In G1, cells integrate environmental and internal signals
that are aimed at the “replicate” / “do not replicate” decision.
S phase is defined by the ability to synthesize genomic DNA. G2
was originally defined as the second gap between S and Mitosis,
but is now known to function as a time of DNA damage repair, and
likely, preparation for entering Mitosis (M phase).
Mitosis has
been traditionally sub-divided into stages defined by nuclear
morphology – prophase, prometaphase, metaphase, anaphase A and
B, and telophase. A final phase, division of the cytoplasm that
overlaps telophase and is often lumped with mitosis, is
cytokinesis (CK).
The major cell
cycle sub-phases, G1, S, G2+M can typically be identified by
direct quantitative measurement of the DNA. With LSC chromatin
condensation measurement can be used to identify a fraction of
the mitotic cells as distinct from G2 cells. The measurement of
additional markers allows for an exploration of the mechanisms
of initiation and control of the cell cycle. This assay explores
the stages of mitosis in great detail. Phospho-S10-histone H3
(pHH3) is used to identify all of the mitotic cells. Cyclin A2,
which regulates S and G2 progression and cyclin B1, which
regulates entry to M and progression from prophase to anaphase
will be used to identify mitotic stages as there expression
levels from maximum to minimum through mitosis.
iGeneration Protocol:
|
Assay
End-point |
Fluorochrome |
Excitation
l |
PMT/PD
Detector |
iGen
End-point |
|
DNA |
DAPI |
405 nm |
Blue:
430–470 nm |
G1, S,
G2, M,
CK |
|
phospho-S10-histone H3 (pHH3) |
Alexa
Fluor 488 |
488 nm |
Green:
515–545 nm |
M |
|
Cyclin A2 |
R-Phycoerythrin
(PE) |
488 nm |
Orange:
565–595 nm |
Progression through Mitotic states |
|
Cyclin B1 |
Alexa
Fluor 647 |
633nm |
Long Red:
650 LP |
iGeneration data*:
*Data courtesy Professor James
Jacobberger, Case Western Reserve University
and Case Comprehensive Cancer
Center, Cleveland OH
Benefits of LSC analysis:
-
Stoichiometric
measurent on a par with Flow Cytometry.
-
Laser excitation,
PMT measurement for high sensitivity, low background and high
dynamic range.
-
Maintain
relationships between stoichiometric measurents and image
data.
-
Individual cell
images as well as “in-context” field images.
-
“Reloacation” from
multivariate analysis to image data.
-
Export data to third
party applications such as Excel (in text format) or FlowJo (FCS
format.)
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