Chromosome advance agglutination specimen preparation experiment
Chromosome advance agglutination specimen preparation experiment
The process of fusing two or more cells of the same or different species into a single cell under natural conditions or by artificial means (physical, chemical or biological) is called cell fusion.
Operation method
basic experiment
Principle
In the early seventies, as a result of the discovery of a certain chromosome-promoting condensation factor in M-phase cells, which is not species-specific, a method for preparing chromosome-advancing condensation specimens was established on the basis of cell fusion and chromosome technology. That is, M-phase cells are allowed to fuse with interphase (I-phase) cells, thus inducing the chromatin of I-phase cells to condense into chromosomes in advance. The chromosomes formed are called Prematurely Condensed Chromosome (PCC). This technique has been applied to cell cycle analysis, the study of the microstructure of chromosomes in normal cells and tumor cells, the study of chromosome damage and repair effects of multiple factors acting on cells, and the clinical practice of predicting the course of a certain blood disease, healing and relapse.
Materials and Instruments
HeLa cells Move 1. Collect cultured M-phase HeLa cells by taking a bottle of HeLa cells in logarithmic growth phase. For more product details, please visit Aladdin Scientific website.
PEG Hanks solution RPMI-1640 medium Calf serum Colchicine KCl Sodium citrate solution Hypomethylenic acid stain PBS Carnoy's fixative Giemsa's stain
Microscope Centrifuge Water Bath Hot Hair Dryer Centrifuge Tubes Needle Syringes Test Tube Racks Staining Tanks Slides Alcohol Lamps
2. Add 10 μg/ml of colchicine to the culture medium to make the final concentration 0.04 μg/ml, and continue to incubate for 12 hours at 37℃ in a carbon dioxide incubator, so that a large number of growing cells were blocked in the M phase.
3. Take a bottle of cells treated as above for each group, and repeatedly shake the bottle in the direction parallel to the growth surface of the cells, so that the culture medium constantly flushed the cell layer (or blowing with a pipette), and the cells in the M-phase can easily detach from the wall of the bottle and be suspended due to their spherical shape.
4. Transfer the medium containing M-phase cells into a centrifuge tube, centrifuge at 1,000 rpm for 5 minutes, discard the supernatant and add 5 ml of Hanks' solution, and blow with a pipette to form a cell suspension.
5. Collect cultured HeLa cells Take a bottle of well-grown HeLa cells and discard the medium.
6. Add a small amount of 0.25% trypsin to digest for 2~3 minutes, discard the trypsin digest.
7. Then add 5 ml of Hanks solution and blow with a pipette to form a single suspension of cells for backup.
8. Preparation of 50% PEG
(1) Weigh 0.5 g of PEG (MW4 000) and pour into a centrifuge tube.
(2) Melt it by heating on an alcohol lamp (about 0.5 ml).
(3) Add 0.5 ml of preheated Hanks solution and mix well, place in a 37°C water bath for use.
9. Cell fusion
(1) Pour the cells from tubes 1 and 2 above into a centrifuge tube and mix thoroughly. centrifuge at 1,000 rpm for 5 minutes, remove the supernatant, and then carefully aspirate the residual liquid.
(2) The cells were dispersed by finger-flick method by aspirating 0.5 ml of 50% PEG under the condition of 37°C water bath and adding it drop by drop into the centrifuge tube with constant gentle shaking, the whole process for about 90 seconds. Then 5 ml of Hanks solution was quickly added to terminate the action of PEG. It was left at 37°C water bath A for 5 minutes.
(3) Centrifuge at 1 000 rpm for 5 min. Discard the supernatant, add 2 ml of RPMI-1640 medium with serum, and at the same time, add 1 drop of 10 μg/ml colchicine vertically with a syringe with a needle tip, gently blow to form a suspension, and incubate in a 37℃ water bath for 30~60 minutes.
10. Preparation of PCC specimens
(1) After the cells were warmed, centrifuged at 1,000 rpm for 5 min to discard the supernatant, and 10 ml of 0.075 mol/L KCl hypotonic solution was added to gently make a suspension.
(2) The cells were treated at 37°C for about 25 min; at termination, 1 ml of Carnoy's fixative was added for fixation, and the supernatant was removed by centrifugation at 1,000 rpm for 8 min.
(3) Finger-flick the bottom of the centrifuge tube to disperse the cells, and add 10 ml Carnoy fixative for fixation for 30 minutes.
(4) Centrifuge at 1,000 rpm for 5 minutes, discard the supernatant and leave 0.2 ml, and gently blow with a pipette to form a suspension.
(5) Take the pre-cooled slide drop a sheet, bake dry, Giemsa staining for about 15 minutes, rinse with water, dry and microscopic examination.
11. Observation of results: Under low magnification, the image of PCC induced by fusion of M-phase and I-phase cells can be easily found. Since cells in different phases of I-phase can all fuse with M-phase cells and be induced to produce PCC, there are three types of prematurely agglutinated chromosomes with different morphological characteristics: G1-phase PCC, S-phase PCC and G2-phase PCC.
(1) The morphological features are:
(1) G1-phase PCC is a single-lineage chromosome, elongated and lightly colored in the form of a fluffy thread mass;
(ii) S-phase PCC has a crushed glume-like structure due to chromosome unraveling and DNA replication at multiple points, with the replicated portion being darker in color and in the form of bilinear chromosome segments;
(iii) G2 phase PCC has condensed bilinear chromosomes because DNA replication is completed, but they are more elongated than M phase chromosomes.