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2S CYTOGEN > U7: KARYOTYPE STAINING PART 2 > Flashcards

U7: KARYOTYPE STAINING PART 2 Flashcards

1
Q

5 Major Steps of Karyotyping

A
  1. Short term lymphocyte culture
  2. Harvesting of lymphocytes
  3. Fixing the cells
  4. Making the chromosome slides
  5. Slide analysis
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2
Q

Short term lymphocyte culture

What component do we need from peripheral blood?

A

Lymphocyte

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3
Q

Short term lymphocyte culture

Collected blood will be grown i__ v_____ by adding mediums.

A

In vitro

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4
Q

Short term lymphocyte culture

What medium/s is/are added to the cell culture?

A
  1. RPMI Growth Medium
  2. Fetal Bovine Serum
  3. Antibiotics
  4. Phytohemagglutinin
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5
Q

Short term lymphocyte culture

What reagent/s is/are used to lyse other cellular components in culture?

A

Hypotonic KCl solution, Glacial acetic acid

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6
Q

Short term lymphocyte culture

Density centrifugation is performed to acquire?

A

Buffy coat

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7
Q

Short term lymphocyte culture

What anticoagulant is used for collection of buffy coat?

A

Sodium Heparin tubes (Green)

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8
Q

Short term lymphocyte culture

This medium is selective for lymphocyte growth.

A

RPMI Growth Medium

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9
Q

Short term lymphocyte culture

Contents of RPMI Growth Medium

A

Amino acids, vitamins, bicarbonate

For the growth of cells

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10
Q

Short term lymphocyte culture

Contents of Fetal Bovine Serum

A

Source of albumin

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11
Q

Short term lymphocyte culture

What is considered a protein overlay?

A

Albumin

Pag maarte daw yung cells, kailangan ng protein overlay - amino acids (whole protein) na ang nasa RPMI and might need albumin

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12
Q

Short term lymphocyte culture

Prevents growth of microorganisms in cell culture

A

Antibiotics

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13
Q

T/F: Lymphocytes in cell culture is only a monolayer, which easily dies in unfavorable conditions.

A

T

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14
Q

Short term lymphocyte culture

What component in RPMI maintains the pH with CO2?

A

Bicarbonate (HCO3-)

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15
Q

Short term lymphocyte culture

This reagent also ensures that only leukocytes are present in the culture, and ensures lymphocyte growth.

A

Phytohemagglutinin (PHA)

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16
Q

Short term lymphocyte culture

How many days should the cell culture be incubated?

A

3 Days

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17
Q

T/F: Cells should be in the logarithmic phase upon harvesting.

A

T

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18
Q

Short term lymphocyte culture

What equipment should you use to check if the cells are in logarithmic phase?

A

Inverted Microscope

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19
Q

Short term lymphocyte culture

This phase is the exponential phase of growth.

A

Logarithmic phase

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20
Q

Short term lymphocyte culture

How many days before harvesting should you split the cell line for significant cell proliferation?

A

2 days

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21
Q

Short term lymphocyte culture

This refers to the first phase in which lymphocytes adjusting to their culture medium.

A

Lag phase

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21
Q

Short term lymphocyte culture

How many days before harvesting should you change the medium?

A

1 day

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22
Q

Short term lymphocyte culture

Specific antibiotics to be used

A

penicillin-streptomycin antibiotics

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23
Q

Step 2 of Karyotyping

A

Harvesting of Lymphocytes

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24
Q

Harvesting of Lymphocytes

This reagent arrests cells at metaphase.

A

Colcemid (Colchicine)

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25
Q

Harvesting of Lymphocytes

How many minutes should you incubate the culture after adding pre-warmed colcemid?

A

15 mins

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26
Q

Harvesting of Lymphocytes

Optimal exposure time to colcemid requires a balance between?

A

proliferative activity index and colcemid concentration

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27
Q

Harvesting of Lymphocytes

This varies whether they are fast replicating or slow replicating

A

Colcemid concentration

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28
Q

Harvesting of Lymphocytes

Cells with high proliferative index requires shorter/longer exposure to a high/low concentration

A

shorter, high

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29
Q

Harvesting of Lymphocytes

Slowly growing cells requires shorter/longer exposure to a high/low concentration

A

longer, low (even overnight)

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30
Q

Harvesting of Lymphocytes

Prolonged exposure to colcemid increases proportion at what stage, and results into?

A

late metaphase, shortening of chromosomes

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31
Q

Harvesting of Lymphocytes

After colcemid treatment, cell pellets undergo what treatment?

A

hypotonic treatment

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32
Q

Harvesting of Lymphocytes

Hypotonic treatment uses?

A
  • KCl solution (0.54%)
  • Sodium Citrate

20 mins at 38C

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33
Q

Harvesting of Lymphocytes

Before hypotonic treatment, the culture must be centrifuged at ____ RPM for how many mins?

A

1000, 10 mins

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34
Q

Harvesting of Lymphocytes

Resuspension in KCl is used to?

A

lyse lymphocytes to isolate nuclear contents

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35
Q

Harvesting of Lymphocytes

After hypotonic treatment, incubate the solution at room temperature for?

A

15 minutes

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36
Q

Harvesting of Lymphocytes

Optional additional modifications that allow for enrichment of long prometaphase chromosomes

A
  • Actinomycin D
  • Ethidium bromide (added before harvesting)
  • Bromodeoxyuridine or BRU (added before colcemid treatment)
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37
Q

Harvesting of Lymphocytes

These are mutagens that interfere with replication

A

Ethidium bromide and BrdU

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38
Q

Harvesting of Lymphocytes

This can significantly increase total yield of metaphase chromosomes

A

Cell synchronization

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39
Q

Harvesting of Lymphocytes

In cell synchronization, cells are arrested at what phase, and how?

A

S phase, add excess amount of BrdU overnight (16 h)

40
Q

Harvesting of Lymphocytes

After excess BrdU. the block is released by?

A

washing the cells and adding thymidine for 5.5 hours before colcemid treatment

41
Q

Step 3 of Karyotype Process

A

Fixing the Cells

42
Q

Fixing the Cells

Cell suspension in hypotonic state must be centrifuged at what RPM and for how many minutes?

A

1200 RPM, 5 minutes

43
Q

T/F: Centrifugation after hypotonic treatment is to remove the cytoplasm and cell membrane of lymphocytes.

A

T

44
Q

Fixing the Cells

Cell pellet will be treated with what fixative solution?

A

Absolute methanol ; glacial acetic acid (3:1)

45
Q

Fixing the Cells

Alternative fixative solution

A

Carnoy’s fixative (ethanol instead of methanol)

46
Q

Fixing the Cells

After fixing, solution will be centrifuged at what RPM and for how many minutes?

A

1200 RPM, 5 minutes

47
Q

Fixing the Cells

Process of fixation and centrifugation is repeated for?

A

3 times

48
Q

Fixing the Cells

In the final addition of fixative solution, it will require incubation at what temperature and how many minutes?

A

4C, 10 mins

49
Q

Step 4 of Karyotype Process

A

Making the Chromosome Slides

50
Q

Making the Chromosome Slides

How many slides will be layered to each other in a paper towel?

A

5 or 6

51
Q

Making the Chromosome Slides

How many drops of the sample will be dropped onto the slide?

A

2 or 3

52
Q

Making the Chromosome Slides

Slides will be stained by?

A

GTG-banding (G-bands by Trypsin using Giemsa)

53
Q

Making the Chromosome Slides

This digests the chromosomes at regions rich in basic amino acids (Arg and Lys)

A

Trypsin

54
Q

Making the Chromosome Slides

Trypsin digests regions rich in what basic amino acids?

A

Arg and Lys

55
Q

Step 5 of Karyotyping Procedure

A

Slide Analysis

56
Q

Slide Analysis

What microscope is used?

A

normal light microscope

57
Q

This is a part of the chromosome which is clearly distinguishable from its adjacent segments.

A

Band

58
Q

How does a band appear?

A

lighter or darker

59
Q

This pertains to a band being more distinguishable from another band

A

Resolution

60
Q

Who published the first paper describing the use of quinacrine mustard?

A

Caspersson et al (1958)

61
Q

The first band staining method

A

quinacrine mustard

62
Q

This is the first attempt to provide nomenclature for chromosome banding

A

Paris report (1971)

63
Q

AT-rich with 2 hyrdogen bonds

A

Heterochromatin

64
Q

GC-rich with 3 hydrogen bonds

A

Euchromatin

65
Q

1971, Summer et al.

A

Giemsa

66
Q

1973, Matsui & Sasaki

A

NOR

67
Q

1978, Linde & Laursen

A

Centromeric

68
Q

Q-Banding

Stain

A

Quinacrine

69
Q

Q-Banding

Microscope Used

A

Fluorescence

70
Q

Q-Banding

Uses and advantages

A
  • ID of all chromosomes and bands
  • revalrs PMPs on Chromosome 3, 4, 13, 14, 15, 21, 22 and Y
  • easily destained for sequential staining
71
Q

G-Banding

Stain

A

Giemsa (GTG), Wrights

72
Q

G-Banding

Microscope used

A

Brightfield microscope

73
Q

G-Banding

Uses and advantages

A

*ID of all chromosomes and bands
* Permanent stain
* Simple photography

74
Q

R-Banding

Stain

A

Giemsa (RHG), CH3/DA

75
Q

R-Banding

Microscope used

A

Brightfield microscope

76
Q

R-Banding

Uses and advantages

A
  • ID of all chromosomes and bands
  • Visualization of ends of chromosomes
  • Small positive R-bands
77
Q

Replication Banding

Stain

A

Hoechst, Giemsa

78
Q

Replication Banding

Microscope used

A

Fluorescence, Brightfield

79
Q

Replication Banding

Uses and advantages

A
  • ID of all chromosomes and bands
  • inactive
  • late-replicating X chromosome
80
Q

C-Banding

Stain

A

Giemsa (CBG)

81
Q

C-Banding

Microscope used

A

Brightfield

82
Q

C-Banding

Uses and advantages

A
  • ID of all centromeric and distal Y heterochromatin
  • reveals polymorphisms including heterochromatic inversions
  • evaluation of ring and dicentric chromosomes
83
Q

NOR Banding

Stain

A

AgNO3

84
Q

NOR Banding

Microscope used

A

Brightfield

85
Q

NOR Banding

Uses and advantages

A
  • ID of active NOR
  • reveals polymorphisms and rearrangements of acrocentric chromosomes
86
Q

DA-DAPI Staining

Stain

A

Distamycin A/DPI and Distamycin A/Hoechst

87
Q

DA-DAPI Staining

Microscope used

A

Fluorescence

88
Q

DA-DAPI Staining

Uses and advantages

A
  • ID of centromeric heterochromatin regions of chromosomes 1, 9, 15, 16, and Y
  • useful in evaluation of chromosomes
89
Q

Giemsa is partnered with?

A

Trypsin

90
Q

Trypsin digests proteins in?

A

Euchromatin

91
Q

T/F: Giemsa only reacts with proteins or sulfate regions.

A

T

92
Q

G-Banding

Lighter stain

A

Euchromatin

transcriptionally active

93
Q

G-Banding

Darker stain

A

Heterochromatin

94
Q

G-Banding

Staining summary

A

AT-rich = darker
GC-rich = light

95
Q

Q-Banding

Staining summary

A

stains AT-rich regions

96
Q

R-banding

Staining summary

A

opposite to G-banding

AT-rich = lighter
GC-rich = darker

97
Q

C-banding

Staining summary

A

stain heterochromatic regions close to centromere

2S CYTOGEN (15 decks)

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