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Applications

Analysis example

Analysis of human peripheral blood lymphocyte subset

Figure1 Dot plot analysis by transmitted light and side scatter (SSC) light
Figure1 Dot plot analysis by transmitted light
and side scatter (SSC) light

Hemolysed human peripheral blood cells were analyzed by PERFLOW. The analysis results of transmitted light (TL)/ side scatter light (SSC) dot plot indicate that transmission signals work as well as forward scatter in measurement of cell size (Figure 1).

Figure2 Dot plot analysis by double staining
Figure2 Dot plot analysis by double staining

The lymphocyte cells were stained using FITC-labeled CD4 antibody and PE-labeled CD8 antibody, and measured by PERFLOW. The CD4-positive T lymphocytes (19%) as well as CD8-positive T lymphocytes (48%) are clearly discriminated in the dot plot analysis (Figure 2).

PERFLOW is expected to contribute to not only the medical research but also the clinical examination of hematological diseases.

Apoptosis analysis

Apoptosis (or programmed cell death) is a normal physiologic process which occurs during embryonic development as well as in maintenence of tissue homeostasis.

figure3

Apoptosis was induced in mouse ES cells and analyzed by PERFLOW after staining with PE Annexin V apoptosis detection kit. The cells that were undergoing apoptosis are shown as PE positive because in the early stage of apoptosis, phosphatidylserine (PS) translocated from the inner to the outer leaflet of the plasm membrane, which bound specifically with PE-conjugated Annexin V. The cells that were in late apoptosis or already dead are both PE and 7-AAD positive since 7-AAD, a DNA specific viability dye became permeable to these cells following the loss of membrane integrity.

  • Living cell:PE(-)/7-AAD(-)
  • Early-stage apoptotic cell:PE(+)/7-AAD(-)
  • Late-stage apoptotic or already dead cell:PE(+)/7-AAD(+)

* Data provided by Dr. Hidemasa Kato, former Associate Professor of Tohoku University Biomedical Engineering Research Organization.

Cell cycle analysis

Cell cycle analysis of HeLa cells stained by PI
Cell cycle analysis of HeLa cells stained by PI

The cell cycle is divided into G0/G1, S, G2 and M phases. The DNA content of G2/M cells (4n) is twice that of G1 cells (2n) because the chromosomes have been doubled during S phase. On the other hand, it is known that polyploid frequently appears in cancer cells, which is shown as more DNA content.
HeLa cells were stained with PI and measured by PERFLOW. The DNA content of each cell cycle phase is shown in the right figure.

Cell sorting examples

Single cell sorting of human iPS cells

It is known that human iPS cell and ES cell are weak to physical damage, and that most of them undergo cell death after sorting of single cells.

Single cell sorted human iPS cell colony
Colony of human iPS cells formed from a single sorted iPS cell

We have for the first time succeeded in generating monoclonal human iPS cells from single cells by using PERFLOW Sort.
The survival rate is more than 60% much higher than the conventional method.
As shown in the photo, sorted single human iPS cell formed a colony after 4 weeks of culture.

Provided by Dr. Tetsuya Ishikawa, General Manager of Cancer Metastasis Laboratory, National Cancer Center Research Institute.

Damage-less sorting of mature megakaryocyte

Mature megakaryocyte is a bone marrow cell with a greatly lobulated nucleus that is responsible for the production of platelets. Due to its size as large as 50~100µm in diameter, it is almost impossible for conventional flow cytometric analysis.

Sorting of megakaryocyte using PERFLOW Sort

PERFLOW Sort has for the first time succeeded in sorting mature megakaryocyte with a high survival rate up to 95%.

State of the cell right after sorting
After sorting Cell looks good
(rupture of the cell is not observed).
State of the cell three days after sorting
State of the cell three days after sorting
The cell is still alive in good condition.
*Provided by Dr. Hiromitsu Nakauchi, Professor of Institute of Medical Science, University of Tokyo

Single sorting of candidate neural crest stem cell derived from transgenic mice

To obtain the candidate neural crest stem cells, the GFP-expressing candidate neural crest cells were isolated from transgenic mouse embryos and single sorted by PERFLOW Sort.

Gene manipulation on fertilized egg of mouse
Gene manipulation on fertilized egg of mouse
Microinjection was performed on a mouse fertilized egg to create a transgenic mouse which specifically expresses GFP in nerve system.
9.5-day embryo
9.5-day embryo
GFP-expressing neural crest cells were isolated from a 9.5-day embryo and single sorted using PERFLOW Sort.
GFP observation
Fluorescent microscopic observation (GFP) of neurosphere
After 20 days of culture, the sorted cell formed a neurosphere.
*Provided by Dr. Harumasa Okamoto, former Department Chief, Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology

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