# Just Flow Cytometry Things (the MACSQuant Analyzer)

rename
omarys's
version from
2017-09-08 18:00

## Introduction

Question | Answer |
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The data from the cytometer can be presented in the form of a dot plot or a ___. | histogram (esp. forward and side scatter data) |

## Some Terminology and Mnemonics

Question | Answer |
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Moving from violet to red, the wavelength of the radiation ___. | increases (UV 400-500-600-700 IR) |

The UV to blue range wavelength is | 355-488 nm |

The blue to green range is | 350-550 nm |

Moiety | each of two parts into which a thing is divided (e.g. in organic chemistry- a functional group) |

Is it too light? Nein! It's heavy. | 2 antibody light-chain isotype and 9 heavy-chain isotypes exist in mammals (genetic variation). |

FITC (fluorescein isothiocyanate) has excitation and emission spectrum peak wavelengths of approximately ___, giving it a ___ color. | 495-530 nm ; green |

Since FITC's ___ maximum is around 530 nm, when we use it we choose ___ filters on our flow cytometers that are centered on a specific region of the spectrum (e.g. 525-30) in order to ___ as many ___ from the fluorescence process as possible. | emission ; bandpass ; capture ; photons |

PE (phycoerythrin), a ___ protein-pigment complex, has an emission maximum of about ___. | red ; 570 nm (but how can it be red if 570 nm is for yellow? check this) |

PE has a more complex ___ state than does FITC; it has two ___ ___: one at around ___ and one at around ___. | excitation ; excitation maxima ; 488 nm ; 561 nm |

Cy5-PE | |

APC | |

GFP | |

PMT | |

Stokes shift: Fluorophores emit photons based upon their emission spectrum, whose maximum is shifted to a USUALLY* ___ wavelength (___ energy) than the maximum of the excitation wavelength. | higher ; (lower) |

*AN EXCEPTION to the Stokes shift phenomenon (i.e. the anti-Stokes process): There is a (albeit very low) probability that the photons emitted from the fluorophore will be ___ the laser excitation wavelength (= will be ___ energy photons). The reason is that the ___ provides some of the ___ itself. | below ; higher ; molecule ; energy |

TorF: The shape of an emission curve is independent of the excitation wavelength. | True. |

## Chapter 1: Here come the FACCI control freaks

Question | Answer |
---|---|

5 techniques help control your flow cytometry experiments and make sure they accurately test your hypotheses. They are: (FACCI - Facsy - Fansy experiment with many controls) | FMO control; Autofluorescence control; Cell counting; Compensation; Isotype control |

NAD(P)H, collagen and riboflavin (cyclic ring compounds), along with amino acids such as tyrosine, tryptophan and phenylalanine (aromatics) have a ___ characteristic, thus requiring ___ ___. | fluorescence ; autofluorescence control |

Autofluorescence results in loss of ___ ___ in certain light ranges, i.e. a ___ in signal ___. | signal resolution ; decrease ; sensitivity |

TorF: Smaller cells typically have more autofluorescence. | False. Larger cells often contain more autofluorescent compounds. |

Isotype control (for the different AB isotypes, which have different binding affinities to cells) can only be used to identify potential ___ problems (i.e. in early development of panels, in revealing ___ issues), but must never be used as the basis of determining ___. | blocking ; (blocking) ; positivity |

Fluorescence Minus One (FMO) control contains all the ___ in a panel except for the one that is being ___. | fluorochromes ; measured |

In an 8-color panel, there would be ___ separate FMO controls. | 8 (each control excludes one of the different fluorochromes) |

The FMO control helps you identify any ___ of fluorochromes into the channel of interest. | spread |

## Chapter 1.5: FACCI control freaks II - Cell counting and compensation

Question | Answer |
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The bottom line of cell counting is that the method you use is ___; you should, however, use a method that is consistent and ___. | unimportant ; reproducible |

It's the gold standard for cell counting; allows for counting cells under a microscope with high accuracy. (Alas, it's not the most efficient method.) | Hemocytometer |

Cell counting via the hemocytometer has some disadvantages, ... | ,,, |

Alternative (automated) cell counting methods can be ___-, ___- or ___-based. | image ; impedance ; cytometry |

Cellometer, T20 and Countess are examples of an- | image-based cell counter |

Image-based counting methods can also count "dead" cells using ___ ___ (similar to what can be done with the hemocytometer). | Trypan Blue |

Coulter, Scepter and Casy are examples of ___-based cell counters. | impedance |

The impedance principle is used for measuring cells in ___. | solution |

The Scepter has the advantage of being a ___, pipet-like device, so it's amenable to ___ cell counting in a ___ ___. | hand-held ; rapid ; tissue hood |

The cytometry-based cell counting principle is basically a simple calculation to determine the ___ of the sample (it provides an accurate measurement of the ___ of sample). | concentration ; (volume) |

Examples of cytometry-based cell ocunters (= of flow cytometers) are- | Accuri and Guava |

A more accurate way than using Trypan Blue for measuring dead cells is to use a- | cell impermeant dye like PI or 7AAD |

For accurate volume measurement (to determine cell count), a ___ ___ must be added to the sample, which is then run on the flow cytometer. | counting particle |

Compensation is the process used to correct ___, the overlap of a fluorochrome into a ___ ___ caused by the physics of fluorescence. | spillover ; second channel |

Manual compensation (= adjusting compensation based on the visual appearance of the data) results in ___ data, which yields ___ conclusions. | overcompensated ; incorrect |

If the (automatic) compensation is to be successful, (1) the controls must be at least as ___ as the samples they'll be applied to. ___ is better, but not off scale. | bright ; Brighter |

If the (automatic) compensation is to be successful, (2) ___ ___ should be the same bwn the negative and positive population. Use of the ___ ___ should be avoided. | background fluorescence ; universal negative |

If the (automatic) compensation is to be successful, (3) the compensation ___ must be the same as the experimental ___ (can't use Alexa488 to compensate for FITC). | color |

## Chapter 2: CReATing an antibody panel

Question | Answer |
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The first step in designing the panel is ___ your antibodies based on ___ ___ level (i.e. on antigen ___) and ___. | ranking ; cellular expression ; (density) ; importance |

Another important step is ___ the antigens to the "right" fluorochrome based on the latter's ___; fluorescence ___ has 2 very important characteristics: ___ power and ___ efficiency. | pairing ; brightness ; brightness ; laser ; detector |

In terms of fluorochrome brightness, you should pair highly expressed antigens such as CD3 with ___ fluorochromes and vice versa. | dimmer |

Instrument configuration: learning the instrument's ___ ___ sources (i.e. the lasers available and their type) and its ___ and ___ control specifications is a significant factor in designing an accurate and effective panel. | excitation light ; sensitivity ; quality |

Lasers can be ___ or parallel; in the former case, some fluorochrome choices may have to be eliminated (i.e. knowing the laser source and the pathways is important for determining the fluorochrome choices that can be used). | colinear |

Sometimes, it is better to use a less ___ fluorochrome if the channel does not receive a lot of ___ (i.e. it's not all about fluorescence intensity). | bright ; error |

In the example of fluorochrome spillover (Excel table), adding the values down the columns results in the amount of ___ a given ___ contributes to the panel, while adding the values ___ results in the error that a given ___ receives from the ___ in the panel. | error ; fluorochrome ; across ; detector ; fluorochromeS |

ABs targeting antigens of low or ___ expression should be paired with brighter fluorochromes. | unknown |

Examples of panel building software include: | Chromocyte ; Fluorish ; FluoroFinder (all can help find the antigen-fluorochrome pairs available/help in the selection process) |

SI: ___ of ABs is important uguise. If too few ABs are used, the sensitivity is reduced (low SI) due to decreased ___ ___. If too many ABs are used, SI also drops, this time because of increased ___. | titration ; positive signal ; background |

SI: Voltage optimization is next (after AB titration). Run a ___ ___ to determine if increasing the voltage will improve the ___. | voltage series ; SI |

This SI formula allows for the ___ of the ___ ___ of fluorochromes. | comparison ; relative brightness |

I compare the differences between the means of the poz and neg and correct this value by dividing by two times the spread (as measured by the SD) of the neg population. What am I? | The SI equation |

Whether you use staining or ___, SI is a critical parameter. | separation (separation index provides a similar metric to the staining index) |

Titration helps save ___ and ___, and avoids ___ due to high concentration of antibodies. | money ; reagents ; background |

OMIP? | Optimized Multicolor Immunofluorescence Panel -- a peer-reviewed, optimized flow panel (the work is already done for you...includes antigens, fluorochromes, analysis template) |

## Chapter 3: Statesticle Anal-ysis

Question | Answer |
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There are two basic classes of questions that are typically asked in flow cytometry: (1) about changes in the ___ or ___ of a specific population on ___ or ___ state. | number ; percent ; treatment ; disease |

Another class of questions asked in flow cytometry is (2) about changes in ___ of a given ___ upon treatment or disease state. | expression ; antigen |

Comparing data is via the T-test, which determines the significance of the ___: define the a value (the ___) and calculate the ___ value. Only if ___ can you reject the null hypothesis and decide that the ___ is statistically significant. | difference ; (threshold) ; P ; P < a ; ??? |

When determining the tendency of expression of a certain antigen (in a "class (2)" question), we use ___ (MFI) and calculate the ___ (rSD); the latter measures the ___ of data around the ___. | median fluorescent intensity ; robust standard deviation ; spread ; median |

Before doing the actual hypothesis testing, it's best to ___ the data to a ___ ___ (_________) value. | convert ; resolution metric (RD) |

The RD is a better form of data because it accounts for the ___ of the data, not just the separation between ___ and ___. | spread ; experimental ; control |

When planning to use the T-test, if you do not have ___ distributed data, there are similar ___ tests that you can perform. They will result in a ___ being reported and identification of statistical significance. | Gaussian ; non-parametric ; P value |

If you have more than two ___/more complex questions, there are additional statistical tools that can be used, e.g. ___ ___ and ___ (ANOVA). | populations ; regression analysis ; analysis of variance |

Using a histogram to illustrate a point after a flow experiment ___ the opportunity to take that ___ data and ___ the analysis into ___ ___. | misses ; content-rich ; extend ; statistical analysis |

the probability of CORRECTLY REJECTING the null hypothesis when the null hypothesis falls = | statistical power |

The power of an experiment is determined by- (3) | the sample size; the spread of the data; the number of replicates |

The power of the experiment, b, is equal to- | True Poz / (True Poz + False Neg) |

The a value is usually ___ and is defined as the chance of finding ___ where there is none (i.e. the chance of committing a ___ error). | 0.05 ; significance ; (type I) |

The advantage of the RD is that issues with the ___ ___ such as ___ and ___ ___ and the instrument can be smoothed out, which makes ___ easier to interpret and understand. | experimental system ; labeling ; cell number ; comparison |

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