Stem Cell Analysis and Enrichment Workshop Flashcards
<p><p>What are 4 ways of determining stem cell expression?</p>
</p>
<p><p>1. Western blotting</p>
<p>2. RT-PCR</p>
<p>3. Immunocytochemistry</p>
<p>4. Flow cytometry</p>
</p>
<p><p>What is RT-PCR?</p></p>
<p><p>Reverse transcriptase PCR. </p></p>
<p><p>How are stem cells identified?</p></p>
<p><p>Stem cells are identified by the expresion or absence of a number of TFs and cell surface markers. </p></p>
<p><p>How can we identify cell populations for enrichment purposes?</p></p>
<p><p>Cross-linking and centrifugation.
MACS
Flow cytometry/FACS</p></p>
<p><p>What is density centrifugation? (words)</p>
| </p>
<p><p>1. Add blood to ficoll-paque (carbohydrate rich solution more dense than water)</p>
<p>2. Centrifuge: RBCs and granulocytes</p>
</p>
<p><p>What is density centrifugation? (picture)</p>
</p>
<p></p>
<p>SSC detector in FC detects what?</p>
<p>Granularity of cells
| FSC: Size of cells.</p>
<p>What does the FSC detector detect?</p>
<p>Size of cells. </p>
<p>What is FACS?</p>
<p>Flourescent-Activated Cell Sorting </p>
<p>How does FACS work?</p>
<p>1. Cell suspension in narrow, rapidly flowing stream of liquid. Flow is arranged so that there is a large separation between cells relative to their diameter.
2. A vibrating mechanism causes the stream of cells to break into individual droplets. The flow passes through a fluorescence measuring station where the flourescent character of each cell of interest is measured.
3. 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 droplet as it breaks from the stream.
4. The charged droplets fall through an electrostatic deflection system that diverts droplets into containuers based upon their charge. </p>
<p>What are the components of a FACS system?</p>
<p>1. A mixed cell suspension - fed into a rapidly flowing stream of liquid.
2. Ability for the flow of the stream to be arranged so that there is a large separation between cells relative to their diameter.
3. A vibrating mechanism which causes the stream of cells to break into individual droplets containing one cell.
4. A laser radiation source and then fluorescence detector linked to a computer.
5. An electrical charged ring around the point at which the stream breaks into droplets which is able to give the droplets either a positive or negative charge based on what the computer tells it to do. Allows sorting of cells into three distinct containers, +ve, -ve or neutral. So three different colours can be read and sorted.
6. An electrostatic deflection system to divert each droplet to its respective container. </p>
<p>What does FACS look like?</p>
What is flow cytometry? [3]
- Method for detecting specific molecules on and within cells.
- Powerful analytical technique for (stem) cell biology research.
- Can be used for sorting and isolating specific sub-populations of cells.
What are the components of flow cytometry ?
A laser source at 90* angle to the flow of single cells.
A SSC detector to detect cell granularity.
A FSC detector to detect cell size.
What are the four main components of flow cytometry?
- A flow cell - liquid stream which carries and aligns cells so that they pass single file through the light beam for sensing.
- A measuring system - commonly used are measurement of impedence and optical systems.
- A detector and analog-to-digital conversion system - which converts analog measurement of forward-scattered light (FSC) and side-scattered light (SSC) as well as dye-specific fluorescence signals into digital signals that can be processed by a binary computer.
- A computer for analysis of the signals.
