Flow Cytometry

• Biocompatibility - the scaffold must be tolerated in the body and it must not produce an adverse immune response.
• Bioactivity - the scaffold must bond to the existing bone.
• Osteogenic - the scaffold must recruit adult stem cells and stimulate them to differentiate into bone producing cells.
• Vascularisation - the scaffold must allow for the development of blood vessels and the supply of nutrients and the removal of waste throughout the scaffold.
• Degradation - the scaffold must degrade at a rate compatible to tissue formation without toxic degradation products.
• Mechanical properties - the scaffold must have similar mechanical properties to the existing bone.

Osteoblasts are bone cells that are responsible for the formation of new bone. Osteoblasts deposit a collagen matrix and release minerals that combine to make the bone mineral.

Flow cytometry is a laser- or impedance-based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second.[pic 1]

2. flow cytometers are able to analyze several thousand particles every second. It can actively separate and isolate particles having specified properties.

A flow cytometer has five main components:

1. a flow cell - liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing
2. a measuring system - commonly used are measurement of impedance (or conductivity) and optical systems (lasers)
3. a detector and Analogue-to-Digital Conversion (ADC) system - which generates forward-scattered light (FSC) and side-scattered light (SSC) as well as fluorescence signals from light into electrical signals
4. an amplification system
5. a computer for analysis of the signals.

Fluorescence-activated cell sorting (FACS)

1. Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous由不同成分形成的 mixture of biological cells into two or more containers, 混合的细胞分开。 one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell.

2. The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets.分成单一细胞一组. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured.分成小滴之前经过fluoresence测量仪  

An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately prior fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream. 分成小滴的地方加电压。根据之前fluoresence的结果，电压不同。

The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. 之后经过一个经典偏离区域，小滴分成两路（根据所带电荷）

Application

The technology has applications in a number of fields, including molecular biology, pathology, immunology, plant biology and marine biology.

1. It has broad application in medicine (especially in transplantation, hematology, tumor immunology and chemotherapy, prenatal diagnosis, genetics and sperm sorting for sex preselection).

2. it is extensively used in research for the detection of DNA damage, caspase cleavage and apoptosis.

3. In marine biology, the autofluorescent properties of photosynthetic plankton can be exploited by flow cytometry in order to characterise abundance and community structure.

4. In protein engineering, flow cytometry is used in conjunction with yeast display and bacterial display to identify cell surface-displayed protein variants with desired properties.

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1 flow cytometry SUWAN

Fluorescence-activated cell sorting (FACS)

1. What is flow cytometry?

Diagram (only the laser part for cell counting)

- In biotechnology, flow cytometry is a laser-based biophysical technology

- in cell counting, cell sorting, biomarker detection and protein engineering

- by suspending cells in a stream of fluid and passing them by an electronic detection apparatus装置.

Components:

1. A flow cell in liquid stream, carries and aligns cells, so that cells pass through sensors one by one, hence have single profile of each cell.
2. A measuring sys, commonly used are measurement of impedance (conductivity) and optical sys (laser)
3. A detector and ADC sys, generates forward-scattering light (FSC) for cell counting and side-scattered light (SSC) for cell sorting to computer and then control the charging voltage, converting the fluorescent signals to electrical signals
4. An amplification sys
5. Computer to analyse signals

1. What is FACS?

Diagram (with fluorescence counters)

- FACS is a specialized type of flow cytometry, in which the cells are labelled by fluorescent probes.

- it provides a method for sorting a heterogeneous mixture of biological cells into 2 or more containers