Mestrado Bioengenharia e Nanossistemas
Projecto Integrado II
António Filipe Sousa, MBioNano
1.1.Flow Cytometry – Definition3
1.2.Principles of Flow Cytometry3
2. Aplications of flow cytometry in stem cell analysis6
3. Stem cell Phenotype characterization using flow cytometry7
3.1 –Human Mesenchymal Stem Cells (hMSC)7
3.1.2. Results and discussion8
3.2. – Mouse Embryonic Stem Cells (mESC)10
3.2.2. Results and discussion11
4. Study of transfection efficiency of hMSC using flow cytometry12
4.1. Transfection of hMSC using lipofectamine and corresponding flow cytometry analysis12
4.1.2. Results and Discussion13
5. Study of human embryonic kidney 293 (HEK293) cells transfection efficiency using flow cytometry analysis13
5.2.2. Results and Discussion15
1.1. Flow Cytometry – Definition
Flow cytometry is a powerful technique for the analysis of multiple parameters of individual cells within heterogeneous populations. Flow cytometers are used in a wide range of applications, such as immunophenotyping, cell counting and reporter gene (e.g. green fluorescence protein (GFP)) expression analysis . This simultaneous parametric model analysis of the physical and/or chemical characteristics is obtained by passing thousands of cells per second through a laser beam and capturing the light of each cell as it passes through it. The data gathered can be analyzed statistically by flow cytometer software to report cellular characteristics such as complexity, size, phenotype or viability, as well to purify populations of interest with Fluorescence-activated cell sorting (FACS) . This technology has a high number of applications, including molecular biology, immunology and in medicine (e.g. transplantation, tumor immunology and chemotherapy, genetics, and sperm sorting for sex pre-selection). The use of fluorescence tagged antibodies is useful in the field of molecular biology as they bind to specific antigens giving unique information about the cells being studied in the cytometer.
1.2. Principles of Flow Cytometry
Fig. 1 - Schematic view of the main components of the Cytometer. 1 – Fluidic system; 2 – Lasers; 3 – Optics; 4 – Detectors; 5 – Electronics and computer system; 6 – interrogation point.
The Flow Cytometer is composed of several components; figure 1 shows a schematic representation of the interior of this equipment. The main components are: the fluidic system, which presents samples to the interrogation point and takes away the waste after that point; the lasers which produce a beam of light of a single wavelength and is directed onto a hydrodynamic focusing stream of fluid; the optic lenses that gather and direct the light; a number of detectors which are aimed at the point where the stream passes through the light beam: one in line with the light beam and several perpendicular to it; and finally the electronics and the peripheral computer system, responsible for the conversion of the electrical signal into digital data and to perform the necessary analyses. The interrogation point is the heart of the system. This is where the laser and the sample intersect and the optics collects the resulting scatter and fluorescence. Fig.2 – Schematic representation of the hydrodynamic focusing in flow cytometer.
For a good data collection, the particles or cells in study must pass through the laser beam one at a time. This is obtained by injecting the sample stream containing the cells into a flowing stream of sheath fluid or saline solution, as represented in figure 2. The sample stream becomes compressed and so narrow that roughly one cell passes through the channel at a time – this is called hydrodynamic focusing....