1. Human Metaphase Chromosomes

2. Experiment Objectives
Preparing
Staining and observing human metaphase chromosomes.

3. Chromosome Morphology
Chromosomes are not visible under the light microscope in non-dividing cells (interphase cells).
As the cell begins to divide, the threads of chromatin (DNA-protein complex) in the nucleus begin to condense into multiple levels of coiled structures recognizable as chromosomes.
There are two modes of cell division:
Mitosis: is responsible for the proliferation of body (somatic) cells,
Meiosis: is responsible for the production of gametes.
Because mitotic cells are easy to obtain, morphological studies are generally based on mitotic metaphase chromosomes.

4. Cell division Cell division can be divided into: Interphase Mitosis
Prophase
Metaphase
Anaphase
Telophase
Cytokinesis

5. Metaphase
At metaphase the chromosomes are at their most condensed state,
Spindle fibers attaching to the area of the centromere called the kinetochore, forming pole-chromosome fibers.

6. Anaphase begins with the division of the centromere and the separation of chromatids.
Once separated, each chromatid is known as a chromosome.
The kinetochore: is the protein structure on chromatids where the spindle fibers attach during cell division to pull sister chromatids apart.

7. Chromosome Analysis
The best mitotic stage for chromosome analysis is pro-metaphase or metaphase.
A typical metaphase chromosome consists of two arms separated by a primary constriction or centromere.
Each of the two sister-chromatids contains a highly coiled double helix of DNA.
Often the sister chromatids are so close to each other that the whole chromosome appears as a single rod-like structure
A chromosome may be characterized by its total length and the position of its centromere.

8. Chromatin and chromosomes are basically the same thing
Chromatin and chromosomes are basically the same thing. The difference is that chromatin is unfolded, uncondensed, extended DNA while chromosomes are condensed DNA. Chromosomes are condensed chromatin and chromatin is unfolded chromosomes.

10. Chromosome Number
The diploid chromosome number is the number of chromosomes in the somatic cell and is designated by the symbol 2N.
The gametes, which have one half the diploid number, have the haploid number N.
Thus, there are 23 pairs of chromosomes in human cells.

11. Of these, 22 pairs are not directly involved in sex determination, and are known as autosomes.
The remaining chromosome pair consists of the sex chromosomes, and is directly involved in sex determination.
In females the two sex chromosom es are identical (XX), whereas in males the two sex chromosomes are not identical (XY).

12. Types of Tissue
A variety of tissue types can be used to obtain chromosome preparations.
Some examples include peripheral blood, bone marrow, amniotic fluid and products of conception.
In the case of blood cell culture only cells that are actively dividing can be used for cytogenetic studies.
Normally only white blood cells are used for cytogenetic analysis.
Specific techniques differ according to the type of tissue used.

13. Overview of Procedure Collection of blood Cell culture
Harvesting: stopping the cell division at metaphase
Hypotonic treatment of red & white blood cells
Fixation
Slide preparation
Staining

14. 1- Collection of blood
Draw 5 ml of venous blood into a sterile heparinized tube containing 0.1 ml of sodium heparin (500 units/ml).
Any other choice of tube is unsuitable since the sample will either arrive clotted or tube will contain EDTA which is toxic to cells and will therefore adversely affect the culture.

15. 2- Cell Culture
Sterile technique must be used throughout the cell culture preparation, because it is possible to cause major contamination during this procedure,
70% of the problems are due to a lack of good sterile technique.
Antibiotics do not eliminate problems of gross contamination which result from poor sterile technique or antibiotic-resistant mutants.
Autoclaving renders pipettes, glassware, and solutions sterile.

16. 2- Cell Culture
Medium
Pipette 10 ml RPMI 1640 medium with L-Glutamine into a 15 ml labeled sterile culture tube
Supplement the medium with the following:
Penicillin-Streptomycin Stock solution
10 µl
( u penicillin/ml – 100 mg/ml Streptomycin
Phytohemagglutinin
0.3 ml
20 µg/ml
Fetal bovine Serum 20%
2 ml
Phytohemagglutinin is A hemagglutinin extracted from a plant - A substance, such as an antibody, that causes agglutination of red blood cells.
has a number of effects on cell metabolism: it induces mitosis,
RPMI-1640 was developed by Moore et. al. at Roswell Park Memorial Institute

17. 2- Cell Culture Incubation
Add 1 ml of whole heparinized blood into the tube containing the supplemented medium
Mix contents of tube with gentle inversion
Incubate in 5% CO2 incubator at 37oC for 72 hours
- CO %, المحافظة على ph للخلايا من الشروط المهمة لنجاح نمو الخلايا بشكل طبيعي

18. 3- Harvesting
Harvesting: mitotic spindle formation is blocked: usually by adding colcemide to the culture, and the cell division is stopped at the metaphase level.
Pre-warm the Colchicine (0.04 mg/ml) in incubator at 37oC.
Add 25 µl of pre-warmed Colchicine to the culture.
Mix gently and incubate at 37oC for minutes.
Note: Colchicine inhibits microtubule polymerization by binding to tubulin, one of the main constituents of microtubules
Availability of tubulin is essential to mitosis, and therefore colchicine effectively functions as a "mitotic poison" or spindle poison
A Tubulin is one of several members of a small family of globular proteins. α-tubulin and β-tubulin, the proteins that make up microtubules
colchicine also inhibits neutrophil motility and activity, leading to a net anti-inflammatory effect

19. 4- Hypotonic treatment of red & white blood cells
Centrifuge for 10 minutes at 2000 rpm.
Discard supernatant without disturbing the cells leaving 0.5 ml of fluid.
Add 1 ml of pre-warmed hypotonic solution (0.075 M KCl) at 37oC.
Mix and then add 9 ml of hypotonic solution.
Mix well by Pasteur pipette.
Incubate at 37oC incubator for 17 minutes,
hypotonic solution should not be in contact with cells more than 27 minutes (may cause rupture of WBCs).

20. 5- Fixation Fixative must be prepared fresh
Add 3 parts of chilled absolute methanol:1 part glacial acetic acid.
Centrifuge for 10 minutes at 1000 – 1500 rpm.
Remove supernatant leaving about 0.5 ml of fluid on top of cells.
At this time there is probably a small whitish or reddish film at the bottom of the tube.
The film contain red blood cell debris and enlarged WBCs.

21. 5- Fixation Add 5 ml of fixative to the tube.
Mix with a Pasteur pipette 3-4 times.
Place in refrigerator for 30 minutes.
Centrifuge the tube for 10 minutes at rpm.
Remove supernatant and add another 6 ml of cold fixative, & mix well.
Repeat the last two steps.
Remove the supernatant leaving 1 ml of fluid at the bottom.
The remaining material will be used to make the slides.

22. 6- Slides Preparation The slide must be exceptionally clean
Lay slides on a paper towel
Withdraw a few drops of cell suspension into a pipette
From a height of 20 cm, drop 2 or 3 drops of fluid on each slide
Allow the slides to dry

23. 7- Staining
Stain the slides by immersion in fresh Giemsa stain for 7-10 minutes
Remove slides from stain & rinse in distilled water
Observe under microscope 40X then under oil immersion

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