L3_4_5_11

L3_4_5_11 - READING FOR TUESDAY Ch 11 p651-660 661-688...

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READING FOR TUESDAY Ch. 11 p651-660, 661-688 Finish techniques Introduction to proteins Introduction to membranes Proteins in membranes First problem set is available on the web
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A flow cytometer has 5 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 2) a measuring system - commonly measurement of impedance (or conductivity) with optical systems - lamps and lasers. Most elaborate 4 lasers and 18 fluorescence detectors 3) a detecting system – takes light into electrical signals 4) an amplification system 5) a computer To clarify, cells can be labeled with up to 18 markers but still can only be sorted into at most 4 streams based on charge: +, ++, -, --. So a cell labeled with label A can be detected and given a negative charge OR a cell labeled with labels A, B, C, D, E, F, G, H etc. can be detected a given a negative charge.
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Fluorescence -activated Cell Sorting can find 1 labeled cell out of 1000. sorts thousands of cells/second A vibrating mechanism causes the stream of cells to break into individual droplets with low probability of more than one cell/droplet http://www.bio.davidson.edu/COURSES/GENOMICS/method/FACS.html
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Figure 3. Quantification of FACS data. This graph compares the number of cells labeled by two colors - red (Y-axis) and green (X-axis). The intensity of the emitted light increases as indicated by the arrows. The number of cells at each intensity is shown by the number of dots where each dot represents a single cell
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Fluorescence Microscopy Fluorescent molecules absorb light at one wavelength and emit it at another-longer wavelength. Uses flters to allow only light o± a given wavelength in and out. Can detect specifc proteins or other molecules in cells and tissues. Fluorescein (emits green), rhodamine (deep red) are molecules that can be chemically coupled to proteins to detect their localization indirectly GFP, isolated ±rom jellyfsh is a protein (encoded by a gene) that has intrinsic ²uorescents Fluorescent molecules can be bleached
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Indirect immunofluorescence allows one to follow the localization of proteins in a cell
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Indirect immunofluorescence
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Section of an E11.5 mouse spinal cord illustrating a gradient of netrin protein (red immunofluorescence) in the path of commissural axons extending to the floor plate at the ventral midline (neurofilament M, green immunofluorescence). Immunofluorescence shows that netrin-1 protein, as distinct from netrin-1 mRNA (see in situ hybridization) is expressed in the floor plate
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RNAi can be used to knock-down the function of genes http://nobelprize.org/nobel_prizes/medicine/laureates/2006/adv.html Fire and Mello 2006 Nobel Double-stranded RNA is cut into short pieces (siRNA) by the endonuclease Dicer. The antisense strand is loaded
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L3_4_5_11 - READING FOR TUESDAY Ch 11 p651-660 661-688...

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