Panel Design in High-Dimension Flow Cytometry

Question 1

What is Spillover spreading matrix for?

Answer 1

Spillover Spreading Matrix (SSM) is for optimal planning design

Question 2

At what point do you need to optimize panel? How do you avoid misinterpreting the data?

Answer 2

10+ colors need more thorough planning to optimize even the low expressing antigen and it is extremely important to have validity and controls to ovoid misinterpretation of data.

Question 3

What does the primary detector do?

Answer 3

The primary detector captures the major emission peak of the fluorochrome

Question 4

What are the potential issues when using a dye?

Answer 4

dye-dye, dye-cell/dye-buffer can occur

Question 5

What can help with the secondary detectors? What is a secondary detector?

Answer 5

Single stained controls are helpful when the secondary detects collect the spillover

Question 6

How does compensation value vary?

Answer 6

compensation value depends on the voltage. It is not a set standard

Question 7

When designing a panel, what do you need to know about the target itself?

Answer 7

when designing a panel, find out the level of the target antigens per cell, co-expression of target markers, and brightness of fluorochrome.

Question 8

what can we help with spreading error? ( think about the system as a whole)

Answer 8

Spillover Spread Matrix provides a way to tackle spreading error in a systematic way

Question 9

What are the 3 key aspects of spreading error

Answer 9

3 key aspects of spreading error
1. Spreading error =signal intensity
2. Spreading error reduces the resolution in the secondary detector
3. Spreading error is additive

Question 10

How can the SSM be calculated?

Answer 10

SSM can be calculated by single cell stained control

Question 11

how to pair a low expressing Ag?

Answer 11

After finding the Ag expression levels, low levels should be in a low spreading error channel with a fluorochrome that has has high spreading error

Question 12

how can spreading error go into designing a panel?

Answer 12

Spreading Error can go into designing a panel in 2 ways
1. Finds fluorochrome pairs with high spillover so assign those two for exclusive markers
2. assess the additive loss of each fluorochrome

Question 13

name some application of flow

Answer 13

Correlate responses and observe changes from treatment, using unbiased analysis of large data sets can detect rare cell type, phenotyping, mouse work.

Question 14

What is experimental workflow

Answer 14

a systematic approach to panel designing

Question 15

Which fluorochrome do you assign to lineage markers

Answer 15

use the fluorochrome with the least spreading error for lineate markers

Question 16

Which detects do you use for dim or unknown targets?

Answer 16

Use detectors with the least amount of spillover for dim or unknown targets.

Question 17

why do technical artifacts occur?

Answer 17

technical artifacts occur, more than likely, to incorrect compensation which is caused by poorly prepared single-stained control

Question 18

how can you see technical artifacts? what are you looking for?

Answer 18

in order to see technical artifacts, inspect the final data of each marker vs. each marker to see common bad pattern of “leaning” triangular population and “super-negative” events

Question 19

How can you diminish artifact effects? what are some issues with it?

Answer 19

correct data transformation (like biexponential, arc sin h , and hyper log display) can diminish artifact effect but optimal transformation depends on the specific data and cannot always be computationally predicted.

Question 20

what can lead to false positive results? how can it be avoided?

Answer 20

dead cells, doublets, or staining artifacts can lead to false-positive results and can be avoided by pre-gating or data cleaning.

Question 21

name some advantages of using high dimension flow cytometry?

Answer 21

thorough panel design allows robust and reproducible flow cytometry with resolution even with dimly expressed markers, save time downstream when designing optimal panel, see rare cell population, and reduce cost from redundant experimental controls.

Question 22

what are some pitfalls of high dimension flow cytometry

Answer 22

Pitfalls mainly occur because of poor planning and lac of controls and suspension need to be single cell so you can’t see your cells interacting with the surrounding environment.

Question 23

what is the best practice of using the best voltage and how to account variability of sample when running on different days

Answer 23

use voltage titration and reference sample

Question 24

What is the problem when using some dyes like BV BUV and Super Bright

Answer 24

you need to use buffers to avoid unspecific binding

Question 25

how do you help the resolution of the primary channel

Answer 25

avoid using colors that overlaps in the secondary channel that interferes the most of the primary channel.

Question 26

how can use see clogs in real time?

Answer 26

designate a histogram plot of SSC (or desired pt. of interest) vs Time to see any irregulates.

Question 27

how should you treat intracellular staining

Answer 27

treat them as experimental sample since there can be a lot of variation with permeability .

Question 28

what should be paired with high expressing Ag?

Answer 28

a fluorochrome with low spreading error or a high spreading channel with a bright signal

Question 29

how important are validation of Ab combination and correct controls?

Answer 29

they are mandatory

Question 30

What do you need with unknown Ag expression level

Answer 30

FMO controls not only for gating but also with spreading error in detectors

Question 31

What can you use unstained for? what are the limits?

Answer 31

autofluorescents but useless in complex experiments

Question 32

what kind of controls are for gating

Answer 32

a biological control or another cell type

Question 33

FMOs cannot do what?

Answer 33

account for unspecific biding, which can cause a shift in the negative population

Question 34

What are isotype controls used for?

Answer 34

staining issues, especially when using secondary antibodies

Question 35

what do you assign bright signals with?

Answer 35

channels with high-spreading error to be above the spread

Question 36

What can cause high spreading error?

Answer 36

signal to a single secondary detector so spreading can improve by avoiding that channel.