Propidium iodide passes through holes in the membranes of dying cells and stains DNA.
Dead cells stain brightly for propidium iodide. We can gate and back gate on our cells. CD25 followed by using the Proliferation tool in FlowJo to break down the generations of blasts from when they were first stained with CFSE. Here we are looking at T cell blasts, stained with CD25 and CFSE. Data can be broken down parameter by parameter with daughter gates and quadrant analysis. Flow cytometry is a very powerful analytical tool because you get both the stats and can visualize your data. Density and contour plots are available in Diva but there are more powerful options in FlowJo. Representative fluorchromes for the red laser. Representative fluorchromes for the blue laser. The set up is light of the longest wavelength is measured first because generally speaking light of longer wavelength has less energy than shorter wavelength light. Notice the long pass filters in front of the band pass filters. Follow the light different colored light, blue is scattered light, red is light generated by the PE-Cy5 label, and the green light was generated by the FITC excitation. Long pass filters let light of a longer wavelength through, short pass shorter wavelengths, band pass second number is +/- N/2. We also see the area of the spectrum that the bandpass filter covers. Here we can see the difference between the excitation (dotted line) spectra and the emission curve for FITC. As more labeled-antibodies or dye molecules bind to the cell, the fluorescent intensity increases. Photoelectric effect (Einstein – Nobel prize, Hertz) Turning up the voltage on a PMT is critical when you expect your sample to be dimly positive. Fluorescent dyes and proteins are important for labeling cells. PE is excited at 488 nm, donates this energy to Cy-7 that emits the energy at 787 nm. The tandem dye PE-Cy7 is excited at 480 565 and emits at 767 nm. PE absorbs at 480 565 nm and emits at 578 nm. The figure to the right is a tandem dye, the first dye catches light of a specific wavelength and transfers the energy to the adjacent molecule. Your excitation wavelength is always shorter than your emission wavelength. We like to use area but we use all parameters to make sure we are looking at single cells. We can set the threshold to gate out noise and small debris. As a cell travels through the laser beam the maximum value attained is when the middle of the cell crosses the middle of the beam. This is an iconic flow cytometry dot plot. Lymphs are much smaller and less granular than monocytes and neutrophils. We can gate out certain cell types using SSC, if you are good you may separate eosinophils from the remaining granulocytes. One lines them up perfectly, the other does not. 
The figure to the right demonstrates the ability of two commercial flow cytometers to discern beads of different diameters. Photodiodes are cheaper and have less sensitivity than PMTs. A photodiode is a type of photo-detector capable of converting light into either current or voltage, depending upon the mode of operation.Focusing lens in front of the FSC photodiode and SSC PMT.The longer the wavelength the lower the energy and vice versa. IR lasers have been used but they are more dangerous to the human eye. Most lasers used for flow cytometry emit light in the visible spectrum.Visible spectrum of electro-magnetic radiation is ~400-700 nm, there are UV and IR lasers.High flow rate (60 ul/min) good for immunophenotyping Low flow rate (12 ul/min) good resolution better for DNA analysis.Laminar flow is when fluids flow in parallel layers and do not disturb each other.Increasing the sample pressure increases the core diameter and therefore the flow rate. The difference in pressure between the sample stream and sheath fluid stream can be used to vary the diameter of the sample core.Lasers specifically semi-conductor lasers have greatly improved. Data is processed in real time on the newest instruments.
Analog to digital converters have made digital instruments possible. Every day there are more mAb, fluorochromes, and stains.IBM analyzers and sorters were provided to the Herzenberg lab who refined the instruments and made the first FACS instruments, BD comes in.This instrument is the ancestor of today’s modern flow cytometers, it lacks the laser and modern electronics but the backbone of a modern cytometer is there.Wallace Coulter’s early patent, cells have more resistance, measure higher voltage.There is no magic but photons, electrons and cells in streams move very fast.
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