1. Human Metaphase Chromosomes

2. Experiment Objectives
Preparing, staining and observing human metaphase chromosomes.
Molecular Biology

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 and meiosis. Mitosis is responsible for the proliferation of body (somatic) cells,
whereas meiosis is responsible for the production of gametes.
Because mitotic cells are easy to obtain, morphological studies are generally based on mitotic metaphase chromosomes.
Molecular Biology

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

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.
Molecular Biology

6. Chromosome Analysis
The best mitotic stage for chromosome analysis is prometaphase 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.
Molecular Biology

7. Chromosome Analysis
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
Molecular Biology

8. Mazen Zaharna Molecular Biology 1/2009

9. 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.
Molecular Biology

10. Overview of Procedure Collection of blood Cell culture
Stopping the cell division at Metaphase
Hypotonic treatment of red & white blood cells
Fixation
Slide preparation
Staining
Molecular Biology

11. 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).
Molecular Biology

12. 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
Molecular Biology

13. 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
Molecular Biology

14. 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
Molecular Biology

15. 3- Stopping cell division at Metaphase
Pre-warm the Colchine (0.04 mg/ml) in incubator at 37oC
Add 25 µl of pre-warmed Colchine to the culture
Mix gently and incubate at 37oC for minutes
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
Molecular Biology

16. 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)
Molecular Biology

17. 5- Fixation Fixative must be prepared fresh
Add 3 parts of chilled absolute methanol: 1 part glacial acetic acid
Molecular Biology

18. 5- Fixation 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
Molecular Biology

19. 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
Molecular Biology

20. 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
Molecular Biology

21. 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 X40 then under oil immersion
Molecular Biology

22. Mazen Zaharna Molecular Biology 1/2009

23. Mazen Zaharna Molecular Biology 1/2009

24. http://www. biology. arizona
Molecular Biology