The graph shows numerical distribution of bone marrow cells related to the DNA concentration.
The cells were stained with a fluorescent dye that binds to DNA.
Using the fluorescence activated cell sorter ( FACS ) the cells were divided according to their different DNA concentration in the nuclei.
Task 1 – questions
Why cells of different DNA concentration are present in the sample?
Why the curve has two peaks?
Is it possible to determine relative duration of the cell cycle phases?
Task 1 – result S 0 number of cells DNA content 1x 2x G1 G2 + M x x
Task 1 - conclusion
Second peak – G2 + M cells – their the DNA concentration is twice higher than this in G1 cells (first peak).
Area between two peaks – S phase cells (they have different DNA concentration, therefore they don't create any peak).
S 0 number of cells G1 G2 + M DNA content 1x 2x
Aneuploidy in Barrett's esophagus Barrett ’ s esophagus = precancerosis Aneuploid cells
Task 2: Observing mouse chromosomes
Slides were prepared from the bone marrow of mouse.
The bone marrow was removed from femur of sacrificed mouse and fixed with the mixture of methanol + glacial acetic acid (3:1).
The suspension of cells was dropped onto the slide and stained with the Giemsa-Romanowski solution (similarly to the human chromosomes in previous practical).
Task 2: How to observe mouse chromosomes
Find the chromosomes or interphase nuclei on the slide using 6x or 10x objective lens.
Change the objective magnification into 40 or 45x and observe chromosomes. Compare the picture in the microscope with adjacent photo.
Enumerate chromosomes in the photo.
How many chromosomes are present in the nuclei of mouse somatic cells?
Describe their shape and determine type of chromosomes.
Task 3: Observing mitosis in onion root tip cells
The root tip is prepared for staining by treating it with 1 M hydrochloric acid, which exposes aldehydes, present in the chromosomal DNA, to which the stain may then attach.
The root tip is stained with Schiff reagent, a dye mixture containing fuchsine that adheres to the aldehyde groups exposed in step 1, thereby coloring the chromosomes and interphase nuclei a deep magenta.
The cells are dispersed into a single layer on a slide, and the slide is prepared for microscopic observation.
Principal of Feulgen ’ s nuclear reaction.
This reaction is a histochemical evidence of the DNA in the nuclei or chromosomes
Scheme of the Feulgen reaction
Task 3: How to observe mitosis in onion root tips?
Find cells on the slide using 6x or 10x objective lens.
Change the objective magnification into 40 x or 45x. Find the area where actively dividing cells are present.
Observe the cells in this area and compare them to adjacent photos until you can easily recognize cells in interphase, prophase, metaphase, anaphase and telophase.
Mitosis in onion root tips
Task 3 – examination of mitotic cells
Using the same high magnification, count the number of cells in each of these phases: prophase, metaphase, anaphase and telophase.
Examine approximately 20-30 of dividing cells. Put down their numbers to the table.
Are portions of cells representing each mitotic phase equal or not? Explain your results.
A table of cells in various mitotic phases 12 4 6 3 Telophase Anaphase Metaphase Prophase Total number Number Phase
Importance of mouse chromosomes examination –mutagenicity (genotoxicity) screening
= genotoxic factor
A chemical, physical or biological agent causing the DNA damage that could result in mutation.
Majority of mutagenic agents have carcinogenic activity (they cause transformation of a normal cell into a tumor one).
Chromosomal changes in two mouse cells after application of genotoxic agent. tetraploidy chromosomal breakage
Task 3 - mitosis
Number of mitotic phases
The share of cells in various mitotic phases is different.
Majority of cells is present in prophase - this phase takes the longest time period of the M-phase.
Mitosis in tumor cells Impairment of cell cycle regulation could result in the malignant transformation of normal cell. Tumor cells have usually high mitotic activity that leads to chromosomal defects (mainly changes of chromosome number).
A ssisted reproduction
Presentation: Ethical issues of assisted reproduction
Test – microscopes, cell cycle, mitosis, DNA structure and its replication
Switch off the microscope lamps an d return slides to boxes, please.
See you next week! Special staining of mouse chromosomes using such called in situ hybridization method.
Fluorescence Activated Cell Sorter
The quantitative measurement of the DNA content of cells was one of the earliest applications in flow cytometry . It has become possible by specific fluorescent dyes which bound to the double helix of nuclear DNA. The fluorescence inside the cell nucleus can easily be detected using this method. Thus, the amount of DNA for cell populations can be studied regarding:
the cell cycle
the ploidy level
Fluorescence activated cell sorter (FACS) measures DNA concentration according to fluorescence intensity. It allows us to measure DNA concentration in cells of patients with hematological and other malignancies.