lec 13: identifying stem cells

surface protein expression

• stem cells express specifc surface proteins (markers)

  • can be.used to isolate, identifty and characterize stem cells

• change during different stages of differentiation and migration

• allow cells to interact and respond to info from their environment

• several surface markers have been identified as cluster of differentiation (CD) and are used to characterize stem cells

flow cytometry

• analyzes single cells as they flow past single or multiple lasers while suspended in a buffered salt-based solution

• determining cell volume, size, and purity of subpopulations which are isolated

• uses lasers to produce both scattered and fluorescent light signals that are read by detectors

• cell populations are analyzed based on their fluorescent or light scattering characteristics

• typical experiment begins with flourescently labeled cells in a single cell suspension

components of flow cytometer

• fluidics system: transporting the sample from the sample tube to the flow cell. once through the flow cell, the sample is either sorted or transported to waste

• optical system: excitation light sources, lenses, and filters to collect and move light around the instrument and the detection system that generates photocurrent

• electronics: the brains → photocurrent from the detector is digitized and processed to be saved for subsequent analysis

foward scatter light signals

• light refracted in the forward direction

• used to determine the relative size of the cell

  • bigger particles produce more than smaller ones, and stronger forward scatter signal

side scatter light signals

• light refracted in a different direction than its original path

• provides information on granularity and complexity of the cells

  • low granularity and complexity → less side scattered light

• more sensitve to membranes, cytoplasm

florescent light emission signals

• detecting the flourescense signal from the conjugated flourophore

• must be careful for autoflourescence → occur from naturally flouroscing substances in the cell

western blot

• detecting certain markers of interest

  1. separate macomolecules in sample using gel electrophoresis

  2. transfered or blotted onto a second matrix, generally a nitrocellulose mebrane → membrane is blocked to prevent any nonspecifc binding of antibodies to the surface of the membrane

  3. transferred protein is then probed with a combination of antibodies:

     1. one specific to the protein of interest (primary antibody)

     2. one specific to the host species of the primary antibody (secondary antibody)

        • complexed with an enzyme, which when combined with an appropriate substrate, will produce a detectable signal

mechanism of detection chemistries

• a detectable signal is generated following binding of an antibody speciic for the protein of interest

• colorimetric → signal is a coloured precipitate

• chemiluminescence: reaction emits light

• flourescence: antibody is labeled with florophore

polymerase chain reaction

• selectively amplify and detect DNA sequences → molecular photocopier

• temp of the sample is repeatedly raised and lowered to help DNA replication enzyme copy the target DNA sequence

  • can produce billion copies of target sequence

3 main steps

1. denaturation of the template DNA into a single strand

2. annealing of primers to each original strand for new stand synthesis

3. extension of the new DNA strand form the primers

   • verifies genetic stability

   • pluripotency marker expression

   • detecting contamination