Download A411-Cell Cycle Assay Kit

Cell Cycle Assay Kit
Catalog # A411
Introduction
Propidium iodide is a kind of the nucleic acid stain. Because propidium iodide intercalates into the
base pair of double-stranded DNA, the fluoresent intensity of a stained cell is directlyproportional
to the DNA content of the cell.Then cell cycle and apoptosis can be analyzed by quantitation of
DNA content with flow cytometry.
Cells in G0/G1 phase contain one set of chromeosome. Supposethe fluorescent intensity of cells in
G0/G1 phase is 1.The fluorescent intensity of cells in G2/M phase which contain two copies of all
chromosomes is 2, and the fluorescent intensity of cells in S phase is between 1 and 2, as cells in S
phase are in the process of DNA replication. For apoptosis cells, nucleus condensation and DNA
fragmentation result in the lost of genomic DNA fragment during the process of PI staining.
Consequently, the fluorescent intensity of apoptosis cell less than 1, and display a sub-G1 peak
which indicate cell apoptosis.
Package Information
component
A411-01 50 rxn
A411-02 100 rxn
RnaseA Solution
PI Staining Solution
1 ml
20 ml
2 ml
40 ml
Storage
Store at -20°C and protect from light.
Assay Protocol
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Collect cells at a density of no more than 2-5×106.
Wash cells twice with cold PBS buffer, and centrifuge the cells at 4°C, 300×g for 5 minutes
each time.
Remove most of the supernate and leave 50-100 μl PBS to resuspend cells gently. Vortex to
achieve a monodisperse cell suspension. Slowly drop 1 ml ice 75% ethanol (precoll at -20°C)
in cell suspension dropwise.
Centrifuge the cells at 4°C, 1000×g for 5 minutes. Wash cells with 1 ml cold PBS.
Resuspend cells with 400μlcold PBS.
Add 20 μl RnaseA Solution to cells suspension and incubate cells at 37°C for 30 minutes.
Filtrate the cells suspension with 400×filter mesh.
Add 400 μl PI Staining Solution, mix gently, incubate cells at 4°C for 30-60 minutes and
protect from light.
Run sample on flow cytometer: set the excitation wavelength at 488 nm.
Precautions
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Cells processing should be gentle, try to avoid artificial damage.
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Because of the washing and fixation steps will cause cell lose, in order to enture the enough
cell for flow cytometry, the initial amount of cells should not be less than 2× 106.Generally ,
the maximun amount of cells harvest from one well of the 6-well culture dishes is 2.5× 106. If
the cells were trated with obvious necrosis, the cells in the supernatant should be collected
together.
During fixation step, it is improtant to maintain the cell suspension, and drop ethanol gently
and dropwise. Add ethanol directly will cause cell agglomerate, and hard to achieve a
monodisperse cell suspension.
After fixation, the cells are hard to be centrifuged, the centrifugal speed should be enhanced
to 1000× g. If the cells still deposit incompletely, he centrifugal speed could be enhanced to
2000× g. Cells after fixation can endure higher speed ofcentrifugation than cells before
fixation.
Filtrate the cells suspension with 400× filter mesh to remove the cell mass and leave the
single cell. If not filtrate, please flip to disperse the cells before staining.
Preparation of tissue: Cut the tissue into small pieces with scissor, digest the tissue with
tryosin for 30- 60 minutes, filtrate the tissue with 200- 400×filter mesh to achieve a
monodisperse cell suspension. Add proper amount of collagenase if the tissue is hard to be
digested.