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omgmbd > L8: Flow Cytometry > Flashcards

L8: Flow Cytometry Flashcards

1
Q

What is flow cytometry?

A

→Measuring properties of cells in flow

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2
Q

What is flow sorting?

A

→Sorting (separating) cells based on properties measured in flow

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3
Q

What is another name for flow sorting?

A

→Fluorescence-Activated Cell Sorting (FACS)

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4
Q

What can a flow cytometry tell us about a cell?

A

→Its Relative Size
→Its Relative Granularity/Internal Complexity
→Its Relative Fluorescence Intensity

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5
Q

What proteins can be used to identify specific cell subsets?

A

→cytokine or chemokine
→metabolic proteins
→adhesion molecules

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6
Q

What are the methods of visualisation?

A

→Fluorescence Microscopy

→Flow Cytometry

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7
Q

What are the differences between the methods of visualisation?

A

→Microscopy- have to scan many fields but FC can analyse thousands of cells.

→FC can detect rare cells.

→FC quantitates the amount of fluorescence in each cell

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8
Q

What can the basics of flow cytometry be divided into?

A

→fluidics
→optics
→electronics

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9
Q

What are the basics of FC?

A 
→Cells in suspension
flow in single-file through
an illuminated volume where they
scatter light and emit fluorescence
that is collected, filtered and
converted to digital values
that are stored on a computer
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10
Q

What are the basic stems of FC?

A

light source→ flow chamber→ optical system→ light detectors→ computer

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11
Q

How are cells suspended in a single file?

A

→by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice
→hydrodynamic focusing

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12
Q

What is hydrodynamic focusing?

A

→Introduction of a large volume into a small volume
→when two streams of fluids with different flow rates are running side-by-side and in the same direction into a flow cell, then a laminar flow is created

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13
Q

What is laminar flow?

A

→Sample fluid flows in a central core that does not mix with the sheath fluid

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14
Q

What is involved in the optics stage of FC?

A

→lasers

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15
Q

Describe lasering of the cells

A

→Single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths
→can provide from milliwatts to watts of light

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16
Q

What sort of frequency is the wavelength of light in the lasers?

A

→Single frequency

→coherent light

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17
Q

Which is more expensive? Air cooled units or water-cooled units?

A

→water-cooled units

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18
Q

What is the meaning of the forward light scatter?

A

→proportional to size

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19
Q

What is the meaning of the side scatter?

A

→granularity

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20
Q

What are dichroic filters?

A

→direct certain wavelengths of light one way, while deflecting other wavelengths

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21
Q

What are PMTs?

A

→amplifies the signal

→photomultiplier tubes

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22
Q

What is involved in the electronics part of FC?

A

→Processing of signals from detectors

→Analog-Digital Conversion

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23
Q

What is Stokes shift?

A

→the energy difference between the lowest energy peak of absorbance and the highest energy of emission

24
Q

What is fluorescence?

A

→absorb light at a particular wavelength and then emit it at a higher wavelength.

25
Q

What are the different fluorochromes and their dyes?

A

→Fluorescein isothiocyanate (FITC)= GREEN

→Phycoerythrin (PE)= ORANGE

→Peridinin Chlorophyll Protein (PerCP) =RED

26
Q

What filters out the overlap between the fluorochromes?

A

→dichroic mirrors

27
Q

Which dye has the highest emission wavelength?

A

→PerCP

28
Q

Which cells are in single cell suspension?

A 
→Peripheral blood
→Bone marrow
→Fine Needle Aspirate
→CSF and other fluids
→Fresh Tissue
29
Q

What are two types of labelling in immunofluorescence?

A

→direct

→indirect

30
Q

What is direct labelling?

A

→Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes

31
Q

What is indirect labelling?

A

→Unconjugated MoAbs
→uses two antibodies.
→Theprimary antibodyis unconjugated and a fluorophore-conjugated secondary antibody directed against the primary antibody is used for detection.

32
Q

What kind of dimension is a histogram?

A

→one dimensional display
→cell number on x-axis, intensity on y-axis
→Only interrogate one fluorescence at a time

33
Q

What is gating in FC analysis?

A

→draw a gate around population of interest and the computer displays only cells in that gate

34
Q

What stain is specific for neutrophils?

A

→CD59 antibody

35
Q

How many populations can be resolved by 3 antibodies?

A

→8

→More antibodies, more flurochromes, more populations can be detected

36
Q

What is propidium iodide?

A

→It undergoes a dramatic increase in fluorescence upon binding DNA

37
Q

What does propidium iodide require for binding?

A

→requires permeabilization of the plasma membrane- punch holes for propidium to get into cells

38
Q

At what wavelength does PI excite and emit?

A

→excite at 500nm

→emit at 600nm

39
Q

How are the number of cells and their content in the cell cycle quantitated?

A

→stained with PI

40
Q

What goes on the x and y-axis of cell cycle quantitation graphs?

A

→X-axis= intensity of fluorescence- proportional to amount of DNA in a cell

→Y-axis= cell count

41
Q

How does PI assay work?

A

→If the PI penetrates the cell membrane, it is assumed to be damaged

→Cells that are brightly fluorescent with the PI are damaged or dead

42
Q

What are the characteristics of apoptosis?

A

→condensation of the chromatin material
→Blebbing of nuclear material
→internucleosomal degradation of DNA
→’ladder’ pattern on DNA gel electrophoresis

43
Q

What are the three ways apoptosis is detected?

A

→staining with the dye PI (cells fixed)
→incubating the cells with fluorescein-labeled Annexin V, and PI
→By staining with 7-aminoactinomycin D

44
Q

Why do apoptosis have ladder appearance on gel electrophoresis?

A

→internucleosomal degradation of DNA

45
Q

Why is phosphatidyl serine(PS) used to detect apoptosis?

A

→normally found on the inside of the membrane

→when cells are apoptosis, it is expressed on the outside.

46
Q

How is PS used to detect apoptosising cells?

A

→incubating the cells with fluorescein-labelled Annexin V, and PI
→cells not fixed

47
Q

What is the difference between using PI staining and annexin V incubation and staining by 7AD?

A

→PI requires cells to be fixed
→ annexin doesn’t require fixing
→7AD- cells not fixed

48
Q

What peak do apoptotic cells display on PI graphs?

A

→sub-50 peak

49
Q

Why are sub-G0 peaks not reliable in detecting apoptotic cells?

A

→peak can be DNA fragmentation.

→Not all apoptotic cells show that peak

50
Q

What are the excite an emission wavelengths of 7AAD?

A

→Ex: ~488 nm

Em: ~660 nm

51
Q

Where does 7AAD intercalate?

A

→ intercalates in G-C regions

52
Q

Which fluorochromes can be used with 7AAD?

A

with FITC & PE labeled Ab for simultaneous evaluation of DNA content and 2-color immunofluorescence using only 488 nm Ex

53
Q

Are live cells negative or positive for 7ADD?

A

→negative

54
Q

What happens to the nozzle tip in cell sorting and why?

A

vibrated so cells break off into droplets containing single cells

55
Q

What happens when the cell of interest gets to the last drop?

A

→charge is applied and pulled by deflection plates into a specific tube

56
Q

What increases after sorting in FC?

A

→Purity has increased after sort

→Allows purification of rare cells

57
Q

What are the applications of flow cytometry?

A 
Immunophenotyping of leukaemias & lymphomas
→Detection of MRD
→Stem cell enumeration
→CD4/CD8 in HIV
→Measurement of intracellular cytokines
→Study of cell cycle, viability & apoptosis
→Measurement of cell proliferation
→Assessment of transfection efficiency

omgmbd (29 decks)

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