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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
Harvesting of Lymphocytes This reagent arrests cells at metaphase.
Colcemid (Colchicine)
25
Harvesting of Lymphocytes How many minutes should you incubate the culture after adding pre-warmed colcemid?
15 mins
26
Harvesting of Lymphocytes Optimal exposure time to colcemid requires a balance between?
proliferative activity index and colcemid concentration
27
Harvesting of Lymphocytes This varies whether they are fast replicating or slow replicating
Colcemid concentration
28
Harvesting of Lymphocytes Cells with high proliferative index requires shorter/longer exposure to a high/low concentration
shorter, high
29
Harvesting of Lymphocytes Slowly growing cells requires shorter/longer exposure to a high/low concentration
longer, low (even overnight)
30
Harvesting of Lymphocytes Prolonged exposure to colcemid increases proportion at what stage, and results into?
late metaphase, shortening of chromosomes
31
Harvesting of Lymphocytes After colcemid treatment, cell pellets undergo what treatment?
hypotonic treatment
32
Harvesting of Lymphocytes Hypotonic treatment uses?
* KCl solution (0.54%) * Sodium Citrate 20 mins at 38C
33
Harvesting of Lymphocytes Before hypotonic treatment, the culture must be centrifuged at ____ RPM for how many mins?
1000, 10 mins
34
Harvesting of Lymphocytes Resuspension in KCl is used to?
lyse lymphocytes to isolate nuclear contents
35
Harvesting of Lymphocytes After hypotonic treatment, incubate the solution at room temperature for?
15 minutes
36
Harvesting of Lymphocytes Optional additional modifications that allow for enrichment of long prometaphase chromosomes
* Actinomycin D * Ethidium bromide (added before harvesting) * Bromodeoxyuridine or BRU (added before colcemid treatment)
37
Harvesting of Lymphocytes These are mutagens that interfere with replication
Ethidium bromide and BrdU
38
Harvesting of Lymphocytes This can significantly increase total yield of metaphase chromosomes
Cell synchronization
39
Harvesting of Lymphocytes In cell synchronization, cells are arrested at what phase, and how?
S phase, add excess amount of BrdU overnight (16 h)
40
Harvesting of Lymphocytes After excess BrdU. the block is released by?
washing the cells and adding thymidine for 5.5 hours before colcemid treatment
41
Step 3 of Karyotype Process
Fixing the Cells
42
Fixing the Cells Cell suspension in hypotonic state must be centrifuged at what RPM and for how many minutes?
1200 RPM, 5 minutes
43
T/F: Centrifugation after hypotonic treatment is to remove the cytoplasm and cell membrane of lymphocytes.
T
44
Fixing the Cells Cell pellet will be treated with what fixative solution?
Absolute methanol ; glacial acetic acid (3:1)
45
Fixing the Cells Alternative fixative solution
Carnoy's fixative (ethanol instead of methanol)
46
Fixing the Cells After fixing, solution will be centrifuged at what RPM and for how many minutes?
1200 RPM, 5 minutes
47
Fixing the Cells Process of fixation and centrifugation is repeated for?
3 times
48
Fixing the Cells In the final addition of fixative solution, it will require incubation at what temperature and how many minutes?
4C, 10 mins
49
Step 4 of Karyotype Process
Making the Chromosome Slides
50
Making the Chromosome Slides How many slides will be layered to each other in a paper towel?
5 or 6
51
Making the Chromosome Slides How many drops of the sample will be dropped onto the slide?
2 or 3
52
Making the Chromosome Slides Slides will be stained by?
GTG-banding (G-bands by Trypsin using Giemsa)
53
Making the Chromosome Slides This digests the chromosomes at regions rich in basic amino acids (Arg and Lys)
Trypsin
54
Making the Chromosome Slides Trypsin digests regions rich in what basic amino acids?
Arg and Lys
55
Step 5 of Karyotyping Procedure
Slide Analysis
56
Slide Analysis What microscope is used?
normal light microscope
57
This is a part of the chromosome which is clearly distinguishable from its adjacent segments.
Band
58
How does a band appear?
lighter or darker
59
This pertains to a band being more distinguishable from another band
Resolution
60
Who published the first paper describing the use of quinacrine mustard?
Caspersson et al (1958)
61
The first band staining method
quinacrine mustard
62
This is the first attempt to provide nomenclature for chromosome banding
Paris report (1971)
63
AT-rich with 2 hyrdogen bonds
Heterochromatin
64
GC-rich with 3 hydrogen bonds
Euchromatin
65
1971, Summer et al.
Giemsa
66
1973, Matsui & Sasaki
NOR
67
1978, Linde & Laursen
Centromeric
68
Q-Banding Stain
Quinacrine
69
Q-Banding Microscope Used
Fluorescence
70
Q-Banding Uses and advantages
* 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
G-Banding Stain
Giemsa (GTG), Wrights
72
G-Banding Microscope used
Brightfield microscope
73
G-Banding Uses and advantages
*ID of all chromosomes and bands * Permanent stain * Simple photography
74
R-Banding Stain
Giemsa (RHG), CH3/DA
75
R-Banding Microscope used
Brightfield microscope
76
R-Banding Uses and advantages
* ID of all chromosomes and bands * Visualization of ends of chromosomes * Small positive R-bands
77
Replication Banding Stain
Hoechst, Giemsa
78
Replication Banding Microscope used
Fluorescence, Brightfield
79
Replication Banding Uses and advantages
* ID of all chromosomes and bands * inactive * late-replicating X chromosome
80
C-Banding Stain
Giemsa (CBG)
81
C-Banding Microscope used
Brightfield
82
C-Banding Uses and advantages
* ID of all centromeric and distal Y heterochromatin * reveals polymorphisms including heterochromatic inversions * evaluation of ring and dicentric chromosomes
83
NOR Banding Stain
AgNO3
84
NOR Banding Microscope used
Brightfield
85
NOR Banding Uses and advantages
* ID of active NOR * reveals polymorphisms and rearrangements of acrocentric chromosomes
86
DA-DAPI Staining Stain
Distamycin A/DPI and Distamycin A/Hoechst
87
DA-DAPI Staining Microscope used
Fluorescence
88
DA-DAPI Staining Uses and advantages
* ID of centromeric heterochromatin regions of chromosomes 1, 9, 15, 16, and Y * useful in evaluation of chromosomes
89
Giemsa is partnered with?
Trypsin
90
Trypsin digests proteins in?
Euchromatin
91
T/F: Giemsa only reacts with proteins or sulfate regions.
T
92
G-Banding Lighter stain
Euchromatin transcriptionally active
93
G-Banding Darker stain
Heterochromatin
94
G-Banding Staining summary
AT-rich = darker GC-rich = light
95
Q-Banding Staining summary
stains AT-rich regions
96
R-banding Staining summary
opposite to G-banding AT-rich = lighter GC-rich = darker
97
C-banding Staining summary
stain heterochromatic regions close to centromere

2S CYTOGEN (16 decks)

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