Fourth seminar- Flow god damn cytometry

Question 1

FLOW CYTOMETRY (FCM)FACS (fluorescence activated cell sorting)
General info-

Answer 1

is a Fast and quantitative laboratory method for multiparametric analysis of single cells.

Flow cytometry (FCM or FACS: fluorescence-activated cell sorting) is a fast and quantitative
laboratory method for multiparametric (physical and chemical properties) analysis of single cells.
Fluorescent probes are used to monitor morphological and functional properties of cells.
The instrument is set to respond to signals derived from the stained cells. Cells are illuminated by laser
beam and the signals coming from them can be detected and analyzed by the photodetectors.

**Flow cytometers have a unique function: these are the only instruments capable of separating a
heterogeneous suspension of cells into purified fractions („sorting”) on the basis of light scattering and
fluorescence properties.

Question 2

The aim of examinations

Answer 2

To detect the absolute and relative amounts of cell subsets
Qualitative and quantitative analysis of pathological cells
Detection of activation state
Cell cycle analysis
Functional characterization

Question 3

Flow cytometry compared to normal microscope…

Answer 3

FLOW CYTOMETRY

10^5-10^6 CELL / EXAMINATION
1-2 MIN/ EXAMINATION
OBJECTIVE (+/-)
GOOD REPRODUCIBILITY
AUTOMATED SAMPLE PREPARATION

MICROSCOPY

10^2-10^3 CELL / EXAMINATION
4-5 MIN/ EXAMINATION
	SUBJECTIVE (+/-)
	POOR REPRODUCIBILITY
	LABOR INTENSIVE SAMPLE PREPARATION

Question 4

ABSOLUTE INDICATIONS OF FLOW CYTOMETRY ROUTINE CLINICAL PRACTICE

Answer 4

HEMATOLOGICAL DISORDERS such as Diagnostics and differential diagnostics of malignant Leukemia recurrence
Minimal residual disease (MRD)
Multidrug resistance (MDR)
Monitoring bone marrow transplantation

and immunodef. such as aids.

Question 5

labeling

Answer 5

either by-

1. By using fluorescence labelled antibodies- Direct or indirect.
2. By using fluorescence dyes binding to organelles specifically

Question 6

filer used in flow cytometry

Answer 6

A dichroic filter is a very accurate color filter used to selectively pass light of a small range of colors while reflecting other colors.

Question 7

What Can a Flow Cytometer Tell Us About a Cell?

Answer 7

Its relative size (Forward Scatter—FSC)

Its relative granularity or internal complexity (Side Scatter—SSC)

Its relative fluorescence intensity
Time-dependency (kinetics) of these parameters

Question 8

Light Scattering properties of cells

Answer 8

Right Angle Light Detector
(Side scatter parameter) Cell Complexity

Forward Light Detector
(Forward scatter parameter)Cell Surface Area

Question 9

Data display- for flow cytometry

Answer 9

The most common ways of depicting flow cytometric data are:
1. histograms to display a single parameter
(usually fluorescence)

Histograms display the intensity of the signals recorded on the x-axis with the number of cells
displayed on the y-axis. Histograms are used to obtain detailed quantitative (percentage) and
qualitative (presence or absence) information about a cell surface or intracellular marker associated
with a defined (gated) cell population

2. dot plots to show distribution of cells in terms of two parameters (e.g. FSC vs. SSc or 2 fluorescent signals).
   “
   Analysis on the basis of size and granularity”

Dot plots are two-parameter data graphs (e.g. FS and SS or FL1 and FL2, etc). Each dot on the
display represents one cell analyzed by the flow cytometer. Dot plots can be used to define cell
subpopulations through displaying co-expression of two different markers (fluorescence signals).

Question 10

Identification of cells by their protein patternIMMUNPHENOTYPING

asked questions:

Answer 10

Immunophenotyping is the analysis of heterogeneous populations of cells for the purpose of identifying the presence and proportions of the various populations of interest. Antibodies are used to identify cells by detecting specific antigens expressed by these cells, which are known as markers. These markers are usually functional membrane proteins involved in cell communication, adhesion, or metabolism. Immunophenotyping using flow cytometry has become the method of choice in identifying and sorting cells within complex populations, for example the analysis of immune cells in a blood sample.

Antigen expression: YES or NO (presence or absence)
Proportion of detected cells
Relative molecule expression
Coexpression

Question 11

INVESTIGATION OF CELL FUNCTIONS

Answer 11

Quantitative measurements of soluble cytokines in biological fluids
Cell cycle analysis
Determining the activation by cell surface and by cytoplasmic markers
Studying phagocytosis
Studying ROI production