Useful knowledge to know Flashcards
When using more than one polymer dye (BUV, BV, BB, SuperBright) in your panel, you need to use a polymer staining buffer (BD Brilliant Stain Buffer or ThermoFisher SuperBright Stain Buffer) to prevent fluorophores from sticking together and causing compensation errors.
NOTE: polymer staining buffers may or may not negatively impact the function of compbeads, so it’s best to just use regular FACS buffer when staining beads.
For unknown reasons, certain fluorophores will have different emission spectra when bound to compbeads vs when bound to cells, and this can lead to poor compensation.
This is common with polymer dyes which work poorly with ThermoFisher’s AbC compbeads, and instead, they work best with UltraComp or UltraComp Plus beads from ThermoFisher.
What are the three rules for good compensation?
- The comp must be at least as bright or brighter than the sample that is being run.
- Background fluorescence should be the same for the positive and the negative controls (beads to beads and cells to cells). (Fixation also alters the autofluorescence, so this also needs to be taken into account).
- Compensation controls must exactly match the experimental fluorochrome and detector settings.
a. Meaning that you must use the same antibody that you stained your samples with, especially when using tandem dyes, and you must also treat your comps the same as your samples (fixing, washes, etc.). This is because the spillover into other channels will be altered by fixation and be different with different antibodies.
One way to reduce spillover when designing panels is to begin with the first detector of each laser. For a 4-color machine, those would be: ?
BV421, FITC, PE, and APC, of which, FITC is the dimmest. PB can be used as an alternative to BV421, but it is dimmer than BV421.
What fluorophores and fluorophore combinations need to be avoided due to spillover?
PE-Cy5 and PE-Cy5.5 should be avoided if possible as these are fluorophores that have large levels of spillover into over detectors.
Combining Percp-Cy5.5 and PE-Dazzle 594 should be avoided.
Combining BV605 and BV650 should also be avoided.
Note that this is what antibody aggregates will look like in flow :) How can you avoid this?
Spin down your antibodies before pipetting. You can also simply gate out antibody aggregates if they ever do appear, but note that the fluorophores needed to gate on to identify aggregates will vary from panel to panel.
Just as a bit of an aside, isotype controls can help identify problems with background staining, but FMOs are superior to isotype controls as FMOs take into account staining errors and spreading error.
Describe the basics of a how a traditional flow cytometer (e.g. BD LSR Fortessa) works.
The cytometer will have different lasers (e.g. yellow-green 561nm laser) that excites certain fluorophores (e.g. PE). Each fluorophore has an excitation spectra (range of light that will excite it [350nm-600nm in figure]) as well as an emission spectra (filled in peak in figure). Filters (e.g. 586/15 as shown by highlighted bar in figure) that capture a part of the emission spectra. The goal of cytometry is to try and choose lasers that excite fluorophores at their peak excitation and use filters that capture their peak emission spectra while limiting other lasers and filters that either excite the fluorophore or capture the emission of the fluorophore when not desired.
CS&T beads are what?
Beads with fluorophores that are a uniform size and fluorescent intensity. They are used for instrument quality control to characterize, track, and report performance of the cytometer.
CS&T = Cytometer Setup and Tracking
What do forward and side scatter measure?
FSC = cell size
SSC = cell complexity/granularity
What are PMT voltages?
Photomultiplier (PMT) voltages are values for each detector on the cytometer that amplifies the signal being picked up by that detector (higher the voltage, the higher the amplification). Since the voltages are simply amplifying signals detected by detectors, they play an important role in allowing for the separation of negative, dimly, and brightly stained cells (CS&T tries to determine the lowest voltage that allows for resolution of dim vs negative cells). However, since it is only amplifying a signal, voltages can NOT change spillover values between different colors as measured by a detector. Instead, panel design will determine the spillover values.
What is the signal to noise ratio in flow?
Signal refers to the relative brightness of the fluorophore being used in combination with the level of expression of the target. Noise is due to electronic background noise of the machine, autofluorescence of the cells, and spillover from other fluorophores.
Explain sensitivity and resolution for flow.
Sensitivity = Can the cytometer detect something that is weakly positive above background noise?
Resolution = How much separation can we get between the negative populations/background and the positive populations.
How do you remove doublets?
Height: Maximum amount of current output by the PMT.
Width: The time interval during which the pulse occurs (measures the time a cell spends in the laser).
Area: The integral of the height and width.
Signal intensity can be measured by either height or area, though area is considered better as it takes into account both height and width.
Single cells and doublets have the same height. However, doublets will have altered width and therefore area, so you can gate out doublets using FSC-A vs FSC-H and/or SSC-A vs SSC-H (Technically could also use width vs area as well).
Number of events you collect for compensation?
5,000 is default, but I’ve been told to do 10,000 for better results.
3 characteristics that define the best PMT voltage?
- Best separation of positive and negative events.
- No events off the top end of the scale.
- Lowest spread of the negative population.
Why increase FSC and SSC voltages for beads vs cells and perm vs non-perm cells?
Beads are smaller, so we need to amplify the voltages of FSC and SSC. Same is true for permeabilized cells where the voltage needs to be increased due to smaller size.
Problems learned from the Haddad lab.
- The rule of 10^3 and above is positive.
- Have to use cells only for compensation/do not treat compensation cells in the same way as the experimental cells.
- Not using Brilliant Stain Buffer when using more than one polymer dye at a time.
Some differences in murine and human cells for flow.
x Humans have several logs higher expression of CD28 (T cell costim marker) compared to mice.
What is compensation?
Compensation refers to the process of correcting for fluorescence spillover, i.e., removing the signal of any given fluorochrome from all detectors except the one devoted to measuring that dye. Thus, compensation does not remove spillover, but it does help correct for it.
What is stain index? (Used in voltage walking)
SI = D/W
D = distance between negative and positive medians
W = robust standard deviation (spread or width of the populations using the median)
With SI, higher is better, so we want max distance and limited spread of populations.
What does compensation being expressed as a percentage mean?
We have run beads only stained for FITC, yet before compensation and because of the overlap of FITC in the PE channel, we see the bead population appear as being both positive for PE and FITC, even though it is only positive for FITC.
And talk some about over/undercompensation.
Through the compensation process, the machine will determine that we need to reduce the PE signal by 30% in the FITC channel so that events with only FITC staining will appear as negatively stained for PE.
Note that, if you are experiencing overcompensate (go over 30% in this example, which leads to swoop down), this could be due to not treating compensation controls the same as the samples (the autofluorescence of the compensation controls doesn’t match samples).
This is also true for undercompensation (going under 30% in this case, which leads to a swoop up), though undercompensation can also be due to using compensation controls that are dimmer than your actual samples.
What is the importance of antigen expression pattern in designing a flow panel?
You may have antigens, like CD4 on T cells, that have a primary expression pattern (Cells either express high levels of CD4 or they don’t), but you may also have other expression patterns:
x Secondary: Cells express a range of densities of the antigen (some high, mid, low).
x Tertiary expression: Antigen expression is either unknown or low.
If an antigen expression pattern is unknown, how do you determine it?
You need to use calibration particles. These are beads with a calibrated number of conjugated fluorochromes that you run with your sample and compare the peaks of the beads and of your sample to determine the antigen denisty.
3 major steps to designing a panel.
Relationship between spillover and resolution?
