Flow cytometry Flashcards
How is flow cytomtry done? State the basics of the instrumentation
It has three parts to it: Fluidics, optics, electronics. Transport cells in fluids in a stream, can change pressure, to the wavelength, they are exposed to forward scatter, seperates them based on size and side sctter- seperates them based on other characteristics. Excited with laser and the wavelength emitted depends on the the fluorochrome type and the optic filter choose the waveband and it’s translate to elctronics where it can be interpeted and data is stored.
How is it analyzed? What factor does accurate analysis depend on?
Using isotypes and antibody- How much? It is displayed on histograms or flow dotplots. It is always compared to a control or a baseline. The voltage has to be consistent, it’s voltage dependent?
Give examples of baselines
They are isotype and concentration matched negative control antibodies
What is gating?
Gating is the filtering of specific cell populations from others using scatter parameters
How do you analyze two different colours?
Dot plots
What are the main questions to ask before analyzing flow cytometry?
Are we trying to see the amount of fluoresence per cell or proportion of positive cells?
How will we gate?
Where is the baseline?
Explain how sorting works?
Can choose sorting criterion when gating, the cells that meet this criteria they are charged with a stream and deflected based on their electric charge between charged deflection plates
What are the common application of flow cytometry?
Phenotyping, apoptosis, live cell assay, monitorig transfections, DNA, cell cycle, signalling, separating
Give example of flow cytometry use in phenotyping cells?
Leukaemia associated phenotype such as CD34 are mature white blood cells but CD13 is myleoid marker
What is proliferation and cell cycle measured with?
DNA labelling fluorochrome like propidium iodide, and BRDU measure S phase
What is a proble with using propidium iodine?
False positives
How does BRDU dye work and how do they measure it?
It binds to thymidine and they use anti-BRDU fluorescence
What other method can measure proliferation? Give example of marker used
Dye dilution, cells are labelled irreversibly with fluorescent dye and with each division the dye will be split and dye will be diluted, know the amount of divisions based on fluorescence. PKH26
What does annexin V bind to?
Exposed phosphtidyl serine on apoptotic cells,
How does pre-G1 staining?
They are fixed with permeabilizing noncrosslinking fixative, treat with RNAse then propidium iodine, dye won’t be able to go inside of cells are alive.
