Flow Cytometry – part 1

Question 1

What can you do with flow cytometry?

Answer 1

• Per cell: measure cell size, granularity and protein expression
• Need cells in suspension

Question 2

How can we quantify different cell types with flow cytometry?

Answer 2

• High throughput
• By morphology
• By protein expression (marker => much more info than just cell type identification)

Question 3

How can you detect markers in flow cytometry?

Answer 3

Marker detection by fluorochrome-conjugated antibodies

=> antibodies that have different targets are bind to different fluorochromes to be able to identify specific cells

Question 4

What does more lasers allow you to do in a flow cytometer?

Answer 4

More lasers = more fluorochromes

With only one laser you are limited to the number of fluorochromes you can detect

Question 5

What can you determine with a forward scatter?

Answer 5

Cell size

Allows to discriminate cell doublets

Question 6

What can you determine with a side scatter?

Answer 6

Cell complexity/granularity

Question 7

What is “gating” in flow cytometry?

Answer 7

GATING: selecting cells of interest (manually)

Question 8

What can be said about absolute cell number counting in flow cytometers?

Answer 8

Standard flow cytometers cannot be used for counting of absolute cell numbers
(except if counting beads are added)

Question 9

What can be said about dead cells in flow cytometry analysis and how can they be excluded (3 ways)?

Answer 9

Dead cells ‘mess up’ your data => non-specific binding of antibodies

1. Gating: dead/dying cells are often smaller
2. DNA-binding dyes: dead cells have permeable cell membranes (get stained)
   - issue 1: are toxic to cells, so longer incubation actually kills cells
   - issue 2: non-fixable
3. amine-binding dyes: more amines inside cell than on surface and dead cells have permeable cell membranes (stain brighter)
   - advantage: less toxic & are fixable

Question 10

What is fluorescence spillover?

Answer 10

Spillover of signal to other detector: you think you’re detecting a black fluorochrome but actually you are still detecting a red fluorochrome

Question 11

How to avoid issues when gating in flow cytometry?

Answer 11

• Avoid square gates

- Use gates that follow a population

Question 12

How can you avoid (when possible) fluorescence spillover (2 ways)?

Answer 12

• Choose fluorophores that have non-overlapping emission spectra
• Use flow cytometers that have multiple lasers (different wavelengths)

Question 13

How can you compensate (if unavoidable) fluorescence spillover (2 ways)?

Answer 13

• Single-stain controls (compensation controls)

- Automated or manual compensation (spillover can often be corrected, but lowers resolution)

Question 14

What can be done to reduce non-specific antibody binding for flow cytometry?

Answer 14

Fc receptor blocking before staining with fluorescent antibodies (incubation with serum or incubation with anti-Fc receptor)

Question 15

What are the factors that influence fluorescence intensity/resolution (3 factors)?

Answer 15

• Choice of fluorochrome: not all are equally bright
• Detector sensitivity (voltage settings)
• Same staining conditions